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I' Michigan State
University

 

This is to certify that the
thesis entitled

CRIMINAL PATERNITY DNA TESTING OF
MlCROSCOPICALLY—IDENTIFIED CHORIONIC VILLI IN
FORMALIN-FIXED PARAFFlN-EMBEDDED
PRODUCTS OF CONCEPTION

presented by

ANN ELIZABETH-CHAMBERLAIN GORDON

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Criminal Justice

 

 

 

 

 

,{x e k
N M 74/ v a

Major Professor’s Signature

5/44/65

Date

MS U is an Affirmative Action/Equal Opportunity Institution

-.~.-.--.—-—.—-c---a-.-—-u—q—-—-c-.-.-.-.—.-.-n-n—u—

- ... .—.-.-c—a----n-.-c-.-—._.—

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/07 p:/C|RC/Date0ueindd-p.1

CRIMINAL PATERNITY DNA TESTING or MICROSCOPICALLY-IDENTIFIED
CHORIONIC VILLI IN FORMALIN-FIXED PARAFFIN-EMBEDDED PRODUCTS
OF CONCEPTION
By

Ann Elizabeth-Chamberlain Gordon

A THESIS

Submitted to
Michigan State University
In partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE
School of Criminal Justice

2006

ABSTRACT
CRIMINAL PATERNITY DNA TESTING OF MICROSCOPICALLY-IDENTIFIED
CHORIONIC VILLI IN FORMALIN-FIXED PARAFFIN-EMBEDDED PRODUCTS
OF CONCEPTION
By
Ann Elizabeth-Chamberlain Gordon

Embryonic/fetal tissue was not easily discerned from maternal decidua in early
abortion materials; therefore, sampling for criminal paternity DNA analysis was
problematic. Microscopic identiﬁcation of chorionic villi (embryonic/fetal placental
tissue) for subsequent DNA testing was pursued. The resultant tissues ﬁom sixteen
early-term elective and spontaneous abortions were examined with no embryonic/fetal
anatomy identiﬁed. Fiﬁy specimens were formalin-ﬁxed, parafﬁn-embedded, sectioned,
stained, mounted on slides, and microscopically examined. Chorionic villi were
identiﬁed for STR DNA analysis. Xylene deparafﬁnisation and tissue lysis buffer
digestion were employed prior to the comparison of two DNA extraction methods—
Chelex® and organic—in recovery of DNA for ampliﬁcation.

Signiﬁcantly higher quantities of DNA and higher quality DNA proﬁle
information (full Proﬁler PlusTM/COﬁlerTM proﬁles) were obtained after Chelex®
extraction. Varying proportions of offspring and maternal alleles—single-source proﬁles
to equal mixtures of contributors—were observed. Discernment of offspring proﬁles,
aided by maternal proﬁle information, for comparison to putative fathers would be
possible for determination of association. Since implementation of the Chelex®

extraction method for this application at the Michigan State Police Lansing Laboratory in

2004, convictions were handed down in ﬁve cases as a direct result of analysis.

To my family

iii

ACKNOWLEDGEMENTS

I would like to thank Dr. Joyce deJong, DO, Medical Director of Forensic
Pathology at Sparrow Hospital, Lansing, M1 for the continued partnership in pursuing
criminal paternity DNA analysis in mid-Michigan and especially in the
implementation of the microscopic identiﬁcation process of chorionic villi. For the
purposes of this research, Dr. deJong prepared the samples from abortion materials
for subsequent DNA testing. Preparation of the tissue included the initial
examination, formalin-ﬁxation, parafﬁn-embedding, sectioning, and microscopic
identiﬁcation of the chorionic villi.

I would also like to thank thesis advisor, Dr. David Foran, PhD, Director—
Forensic Science Program, School of Criminal Justice and Department of Zoology,
Michigan State University for approving the proposed research, guiding the
methodology and manipulation of data, and painstakingly overseeing the writing of
this research paper. Thank you for the encouragement and support especially in the
ﬁnal stages of project completion.

Thank you to Dr. Christina DeJong Schweitzer, PhD, Associate Professor,
School of Criminal Justice, Michigan State University for guiding the statistical
portions of this research. The patience, effort, and accessibility afforded were greatly
appreciated.

Thank you to the Michigan Department of State Police Forensic Science
Division for moral support and encouragement, ﬁnancial support for reagents, and

access to all necessary equipment.

iv

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF TABLES viii
LIST OF FIGURES x
INTRODUCTION 1
Rape Offenses in Michigan 1
Occurrence and Reporting of Rape 2
Transfer of Biological Evidence in Rape Cases 2
Collection of Embryonic or Fetal Materials for DNA Analysis ------------- 3
Abortion Techniques and Possible Effects on Sample Collection,
Preservation, and DNA Testing 3
Initial Screening and Gestational Age Determination 3
Abortion Techniques 4
Evidence Handling of Abortion Materials in Mid-Michigan
Prior to 2002 5
Evidence Handling of Abortion Materials in Mid-Michigan from
2002 to Early 2003 5
Evidence Handling of Abortion Materials in Mid-Michigan in
Early 2003 6
Choriom'c Villi Development 6
Forensic STR Analysis 12
Factors Affecting DNA Recovery from Tissue Samples 15
Organic and Chelex® 100 Extraction of DNA 15
Limitations in Interpretation of Genetic Testing Results 16
Goal of this Research 18
MATERIALS AND METHODS 19
Institutional Review Board (IRB) Approval 19
Sample Collection and Preparation 19
Fixation 19
Tissue Processing 20
Sectioning 20
Staining 20
Microscopic Examination 21
Preparation of Parafﬁn-Embedded Embryonic or Fetal Tissue and
Maternal Decidua for DNA Extraction 21
Sample Preparation 21
Deparafﬁnisation 22
Digestion of Tissue, Puriﬁcation of DNA and Concentration
of DNA 23

 

RESULTS

DISCUSSION

DNA Extraction of Embryonic or Feta] Tissue and Maternal
Decidua

 

Chelex® Extraction

 

Organic (phenol/chloroform/isoamyl alcohol) Extraction ------

Quantiﬁcation of DNA

 

DNA Ampliﬁcation

 

Sample Preparation for Electrophoresis and GeneScan Analysis -------

 

Data Interpretation

Determination of Full vs. Partial Single-Source Proﬁles --------

 

 

 

 

 

 

 

23
24
24
25
25
26
27
27

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Determination of Full vs. Partial Mixture Proﬁles 28
Mixture Allele Relationships Representative of the Mother
and a Full Offspring 28
Sample Concordance 28
Statistical Analyses 29
32
Parafﬁn Extraction 32
DNA Quantiﬁcation 32
Ampliﬁcation of Electrophoresis of Puriﬁed DNA 42
TS 1—Proﬁle Results 46
TSZ—Proﬁle Results 47
TS3—Proﬁle Results 47
Genetic Proﬁle Composition 48
Chelex®-Extracted and Proﬁler PlusTM-Ampliﬁed Samples 55
Chelex®-Extracted and COﬁlerTM-Ampliﬁed Samples 58
Organically-Extracted and Proﬁler PlusTM-Ampliﬁed Samples --------- 59
Organically-Extracted and COﬁlerTM-Ampliﬁed Samples 60
Concordance of Proﬁles Between Ampliﬁcation Systems and
Extraction Methods 60
Identiﬁcation of Associated Samples 63
Comparison of Single-Source Female Proﬁles to Associated
Samples 65
Peak Height Imbalance Between Loci 66
Off Ladder Alleles 70
Statistical Estimates 73
75
Historical Issues and Procedures Developed 75
DNA Recovery and Isolation of Embryonic/Fetal DNA 76
Concordance of Samples Between Ampliﬁcation Systems and
Extraction Methods 78
Identiﬁcation of Associated Samples 79
Explanation of Superior Chelex® Results 79
Data Anomaly Observed 82

APPENDICES

REFERENCES

Implementation of Chelex® Extraction Method for Embryonic/Fetal

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tissue in the Forensic Laboratory 84
Case 1 85
Case 2 86
Case 3 88
Case 4 90
Case 5 92
Case 6 93
Recommendations for Handling Aborted Materials in Criminal
Cases 94
Conclusions 96
98
APPENDIX A STATUTORY CIRCUMSTANCES FOR CSC I
and CSC II 99
APPENDIX B STATUTORY CIRCUMSTANCES FOR
CSC III and CSC IV 102
APPENDIX C PROTOCOL FOR THE PREPARATION OF
FORMALIN-FIXED PARAFFIN-EMBEDDED
EMBRYONIC/FETAL TISSUE AND MATERNAL DECIDUA
FOR DNA EXTRACTION 104
Sample Preparation 105
Deparafﬁnisation 105
Digestion 105
Collection and Puriﬁcation 106
APPENDIX D PROTOCOL FOR CHELEX® EXTRACTION
OF DNA 107
Chelex® Extraction Protocol 108
APPENDIX E PROTOCOL FOR PHENOL/CHLOROFORM
/ISOAMY L ALCOHOL (ORGANIC) EXTRACTION OF
DNA 109
Organic Extraction Protocol 110
111

 

REFERENCES

 

vii

112

LIST OF TABLES

TABLE l—Quantiblot results of human DNA recovered from Chelex®

33

 

extraction of Test Set 1 samples

TABLE 2—Quantiblot results of human DNA recovered from Chelex®

34

 

extraction of Test Set 2 samples

TABLE 3—Quantiblot results of human DNA recovered from Chelex®

35

 

extraction of Test Set 3 samples

TABLE 4—Quantiblot results of human DNA recovered from organic

 

extraction of Test Set 1 samples

TABLE S—Quantiblot results of human DNA recovered from organic

36

 

extraction of Test Set 2 samples

TABLE 6—Quantiblot results of human DNA recovered from organic

37

 

extraction of Test Set 3 samples

TABLE 7——Full genetic proﬁles generated for Test Set 1 samples

 

TABLE 8—Full genetic proﬁles generated for Test Set 2 samples

 

TABLE 9—--Full genetic proﬁles generated for Test Set 3 samples

56

 

TABLE 10—Genetic proﬁle composition of Test Set 1 samples

 

TABLE 11—Genetic proﬁle composition of Test Set 2 samples

57

58

 

TABLE lZ—Genetic proﬁle composition of Test Set 3 samples

TABLE 13—Associated samples from Test Sets 1, 2, and 3

63

 

85

 

TABLE 14A—Genetic data at 13 CODIS core loci for Case 1

TABLE l4B—Genetic data at 13 CODIS core loci for Case 1

86

 

 

TABLE ISA—Genetic data at 13 CODIS core loci for Case 2

TABLE 15B——Genetic data at 13 CODIS core loci for Case 2

87

88

 

TABLE 16A—Genetic data at 13 CODIS core loci for Case 3

89

 

viii

TABLE 16B—Genetic data at 13 CODIS core loci for Case 3

 

TABLE 17A—Genetic data at 13 CODIS core loci for Case 4

90

91

 

 

TABLE 17B—Genetic data at 13 CODIS core loci for Case 4

TABLE 18—Genetic data at 13 CODIS core loci for Case 5

 

TABLE 19—Genetic data at 13 CODIS core loci for Case 6

 

92

93

94

FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

LIST OF FIGURES

1—10X view of chorionic villi from the microscope slide prepared from
sections of tissue block sample TS3-L 8

 

2—Primary chorionic villi surrounded by a thin layer of mesoderm
(connective tissue) 9

 

3—Secondary chorionic villi surround by a thin layer of mesoderm
(connective tissue) 10

 

4—Tertiary villi and intervillous spaces facilitate placental circulation --------- 11

5— Photograph of an embryo at 7 weeks contained within the chorionic
sac 12

 

 

6—Photo graph of the parafﬁn block containing sample TS 1 -4A 22

7—Comparison of DNA recovery from TS-l Chelex®- and organically-
extracted samples 39

 

8—Comparison of DNA recovery ﬁ'om TS-2 Chelex®- and organically-
extracted samples 40

 

9—Comparison of DNA recovery from TS-3 Chelex®- and organically-
extracted samples 41

 

10—Number of full proﬁles generated for Chelex® extraction and organic
extraction 43

 

ll—Sample TSl-l (extracted with Chelex® and ampliﬁed with the Proﬁler
PlusTM ampliﬁcation kit) exhibited a full single-source female proﬁle----49

12—Sample TS1-7 (extracted with Chelex® and ampliﬁed with the Proﬁler
PlusTM ampliﬁcation kit) exhibited a full single-source female with
additional activity proﬁle 50

 

13—Sample TSl-6 (extracted with Chelex® and ampliﬁed with the Proﬁler
PlusTM ampliﬁcation kit) exhibited a single-source male proﬁle ----------- 51

14—Sample TSl-S (extracted with Chelex® and ampliﬁed with the Proﬁler
PlusTM ampliﬁcation kit) exhibited a single-source male proﬁle with

additional activity 52

 

FIG. 15—Sample TSZ-ll (extracted with Chelex® and ampliﬁed with the Proﬁler
PlusTM ampliﬁcation kit) exhibited a mixture of female DNA ------------- 53

FIG. 16—Sample TS2-6 (extracted with Chelex® and ampliﬁed with the Proﬁler
PlusTM ampliﬁcation kit) exhibited a mixture of female and male DNA--54

FIG. 17—Sample TSl-7 (extracted organically and ampliﬁed with the Proﬁler
PlusTM ampliﬁcation kit) exhibited a mixture of female and male DNA
(D3Sl358, amelogenin, D8S1179) 62

 

FIG. 18—Sample TS2-10 (extracted with Chelex® and ampliﬁed with the Proﬁler
PlusTM ampliﬁcation kit) exhibited a single-source female proﬁle with
additional activity 64

 

FIG. 19—Sample TS1-4B extracted with the Chelex® method (ampliﬁed with the
Proﬁler PlusTM ampliﬁcation kit) 68

 

FIG. 20—Sample TS1-4B extracted with the organic method (ampliﬁed with the
Proﬁler PlusTM ampliﬁcation kit) 69

 

FIG. 21—Sample TS2-3 (organically-extracted) with OL allele designations -------- 71

FIG. 22—Sample TS2-3 (organically-extracted) with allele designations ------------- 72

 

FIG. 23—List of recommendations for law enforcement personnel 95

xi

 

INTRODUCTION

Rape Offenses in Michigan

Michigan law outlines in detail what constitutes rape or criminal sexual conduct
(CSC). CSC offenses are assigned one of four degrees. First and third degree CSC
offenses (CSC I and CSC III) involve penetration. Second and fourth degree offenses
(CSC II and CSCIV) involve contact in a sexual manner. The degree of a CSC offense
must be accompanied by at least one statutory circumstance. Some of the statutory
circumstances that apply to CSC I and CSC II are as follows: the victim is under 13 years
of age, the victim is at least 13 but less than 16 and the perpetrator is a member of the
victim’s household or related by blood or in. a position of authority over the victim, the
sexual act involves commission of any other felony, and the perpetrator is armed with a
weapon or an article fashioned so as to lead a person to reasonably believe it is a weapon.
A different set of statutory circumstances apply to CSC III and CSC IV. Some of these
are as follows: the victim is at least 13 years of age but less than 16 (CSC 111 only), the
victim is at least 13 but less than 16 years of age and the perpetrator is ﬁve or more years
older than the victim (CSC IV), the perpetrator uses force, and perpetrator knows or has
reason to know the victim is mentally incapable, mentally incapacitated, or physically
helpless. Complete lists of statutory circumstances can be found in Appendix A and
Appendix B.

Statutory rape (non-forcible intercourse with or between individuals, which would
otherwise be legal, if not for their ages) differs from forcible rape in the matter of

consent. Intercourse with a minor is illegal in all 50 states, but the age of consent varies.

In Michigan, as previously described, any sexual intercourse, even non-forcible, with an
individual under the age of 16 is illegal and prosecutable.

Occurrence and Reporting of Rape

 

For a variety of reasons, females do not always report sexual crimes in a timely
manner, or at all. Some of these include the desire to avoid embarrassment, fear of
further harm (Kilpatrick et al., 1992; Kilpatrick, 2000), a statutory rape situation, or
forcible rape of a minor in which discovery was prevented by the perpetrator and/or the
circumstances. All of the aforementioned can make evidence recovery difﬁcult or
impossible.

T_ransfer of Biological Evidence in Rape Cases

Physical evidence of interest in the prosecution of rape or criminal sexual conduct
cases can vary with the circumstances. The best evidence is the direct transfer of bodily
ﬂuids (vaginal secretions, semen, seminal ﬂuid, saliva, or perspiration containing
epithelial cells) between the victim and perpetrator during intercourse and the transfer of
ﬂuids to the environment or to a condom. In the crime lab, bodily ﬂuids are located,
identiﬁed, isolated, and individualized using DNA analysis methods. The results are
compared and the evidence can be declared a match to the exemplar, or the exemplar can
be excluded as a possible donor to the evidence.

A small percentage of the time a victim becomes pregnant as a result of the
assault, and in some of these cases, the pregnancy is the only evidence that a forcible or
statutory rape occurred. The Rape, Abuse, and Incest National Network (RAINN) (2006)
calculated that out of the 247,730 rapes that occurred in 2002, approximately 4,315

pregnancies (~1.7% of reported rapes) resulted. The calculation is based on information

from the US. Department of Justice, Bureau of Justice Statistics’ 2002 National Crime
Victimization Survey and medical reports.
Collection of Embryonic, Fetal, and Full-Tenn Offspring Samples for DNA Analysis

If a pregnancy resulting from rape is not aborted, a sample can be obtained from
the embryo, fetus or the full-terrn baby and compared using paternity DNA analysis with
the victim and suspect samples. Samples of chorionic villi—tiny outgrowths ﬁ'om the
outer membrane chorion surrounding an embryo that grow into the womb wall and help
to form the placenta (see below)——can be obtained during gestation (Lobbiani et al.,
1991; Karger et al., 2001; Mingjun et al., 1993; Reshef et al., 1999). After birth, a
sample can be obtained by swabbing the infant’s mouth or by drawing a whole blood
sample for analysis. If the mother decides to terminate the pregnancy, DNA analysis of
the embryonic or fetal tissue obtained during an abortion procedure is possible. The
present study focused on the viability of the latter method.
Abortion Techniques and Possible Effects on Sample Collection, Preservation, and DNA
Titing
Initial Screening and Gestational Age Determination

The ﬁrst step in the abortion process is the actual conﬁrmation of pregnancy
(Smith, 1982). The pregnancy hormone human chorionic gonadotropin (hCG) can be
detected approximately two weeks after fertilization or four weeks since the last
menstrual period. A positive result from a simple 5-minute urine test is an indication of
pregnancy. Ultrasound and pelvic examination will attempt to conﬁrm pregnancy and to
identify growth stage (Smith, 1982). Many women have irregular menses or have

experienced bleeding that was interpreted as menses, which can throw off the estimation

of gestational age (Hem, 1984). Documentation of the gestational age of the pregnancy
at the time of abortion is important to law enforcement, as well as pathology and forensic
laboratory scientists. Once the gestational age is determined, the appropriate abortion
procedure can be performed.

Abortion Techniques

According to the World Health Organization (2003), the vacuum aspiration
technique generally performed through 12 weeks utilizes a vacuum, either manual or
electric, to empty the uterine contents. An aspirator or syringe is connected to a cannula
ranging in size from 4—12 mm in diameter which is connected to the vacuum source.
Dilation of the cervix may be necessary for insertion of the cannula and/or syringe. The
vacuum process takes 3—1 0 minutes and is performed under local anesthesia or
analgesics. During the vacuum process, the tissues may suffer gross trauma, distorting
them beyond recognition (Karger et al., 2001 ), making it difﬁcult or impossible to
identify fetal components for DNA analysis.

Another procedure, dilatation and curettage (D&C) is used through the 12th week
of pregnancy. This involves the dilation of the cervix with mechanical dilators or
pharmacological agents and the use of sharp metal curettes to remove tissue from the
walls of the uterus. The procedure may also distort the tissue due to gross trauma
(Karger et al., 2001). The dilatation and evacuation (D&E) procedure is used after the
12th week of pregnancy until the 23rd week. The cervix is dilated and the uterus is
evacuated using a vacuum and a 14—1 6 mm diameter cannula and forceps. The dilation
procedure may take 2 hours to one full day. An alternative to the aforementioned

procedures is the chemical induction method. It utilizes an anti-progesterone drug to

interfere with the continuation of the pregnancy, and a prostaglandin to enhance uterine
contractions to expel the products of conception. This method is far less destructive to
tissues.
Evidence Handling of Abortion Materials in Mid-Michigan Prior to 2002

Prior to 2002, the aborted embryo or fetus (resultant from rape) and the maternal
materials were frozen or ﬁxed in formalin and shipped directly to the Michigan State
Police Lansing Laboratory for analysis at any stage of development. Well-developed,
whole fetuses were oﬁen received even though swabs of the mouth or a blood sample
could easily have been collected—causing adverse psychological stress to the analyst, as
well as storage and disposal problems. Materials from very early abortions
(approximately 2—7 weeks post-conception) exhibiting amorphous tissue were also
frequently received. Identiﬁcation of embryonic or fetal tissue for DNA analysis relied
on visual recognition of fetal anatomy within or among the materials. According to
Moore and Persaud (1993), the embryo or fetus may not be readily visible to the naked
eye before the 8th or 9th week. This is especially true if the anatomy was badly distorted
due to the abortion process (Karger et al., 2001). Due to the difﬁculty in visual
identiﬁcation of the embryonic or early fetal parts in early abortions, random samples
from the materials would be used for genetic testing and often results were limited to the
maternal proﬁle.
Evidence Handling of Abortion Materials in Mid-Michigan from 2002 to Early 2003

Difﬁculties with storage, disposal, and analyst stress provoked an agreement
between Dr. Joyce deJong, Medical Director of Forensic Pathology at Sparrow Hospital

in Lansing, Michigan and the Michigan State Police Lansing Laboratory Biology Unit,

with regards to processing of abortion materials. Forensic Pathology received all aborted
material cases and screened them for embryonic/fetal anatomy. If such anatomy was
located, samples were collected and forwarded to the Michigan State Police Lansing
Laboratory. If anatomy was not located, it was agreed that the case would not be pursued

for DNA analysis.

Evidence Handling of Abortion Materials in Mid-Michigan in Early 2003

 

In early 2003, a study conducted by Karger et al. (2001) was discovered, which
discussed a procedure to identify very early embryonic tissue structures, chorionic villi,
for forensic genetic testing. An agreement was reached between Dr. deJong and the
Michigan State Police Lansing Laboratory to conduct further research on the topic and to
attempt implementation. This procedure suggested formalin-ﬁxation, parafﬁn-
embedding, sectioning, and hematoxylin-eosin staining of early-term abortion materials.
DNA analysis would be conducted on sections demonstrating the presence of chorionic
villi.

Chorionic Villi Development

The stages of embryonic and fetal development are important in microscopically
identifying chorionic villi—cells ﬁom the fetal side of the placenta (Fig. 1). According
to Moore and Persaud (1993), the primitive chorionic villi appear by the end of the 2nd
week following conception. The embryonic period begins at the beginning of the 3rd
week just after the primitive chorionic villi appear. The primary chorionic villi (Fig. 2)
begin to branch, and in several days they cover the entire chorionic sac becoming
secondary chorionic villi (Fig. 3). Within a few days the venous networks are present,

and the structures become tertiary chorionic villi or stem villi (Fig. 4). Blood begins to

ﬂow through the villi by the end of the 3rd week. The limb buds start to develop at the
end of week four and beginning of week ﬁve. At the end of week ﬁve, the embryo is
often still too small (8 mm) to identify with the naked eye. By the beginning of week six
distinct ﬁngers are beginning to form, and if left intact the embryo (13 mm) may be
visually identiﬁed at the end of this week (Fig. 5). The embryo will grow to be
approximately 30 mm by the end of the 8th week, when it can frequently be identiﬁed
visually. During the 9th week, the fetus is easily identiﬁed visually as it is 50 mm in
length. To identify the chorionic villi from an early aborted pregnancy, the maternal
decidua and/or embryonic/fetal material must be chemically ﬁxed, embedded in parafﬁn,
sectioned, stained, and mounted. Microscopic evaluation identiﬁes slides containing

chorionic villi.

 

FIG. l—Microscopic cross-section of chorionic villi and maternal tissues. Three
layers of tissue, chorionic villi which are ﬁnger-like in appearance with lacunae (empty
spaces that would contain maternal blood), maternal endometrium (spent glands exhibit
saw-tooth edges—to the immediate left of the identifier), and myometrium (maternal) are
represented from the top down (Duker, 2003).

Uterine vessels Uterine glands

   
  

Syncytiotrophoblast

Cytotrophoblast

., .. .1»
."o'fm. .II"~‘ r:
grin-rant)? ~'

 

 

Mesoderm Intervillus space (lacunae)

 

FIG. 2—Primary chorionic villi surrounded by a thin layer of mesoderm (connective
tissue). The mesoderm is covered by cytotrophoblast and superﬁcially with
syncytiotrophoblast (cells that contact maternal blood) (Gray, 2000 (1918)).

Uterine glands Uterine vessels

  
 

Syncytiotrophoblast

Cytotrophoblast

Core of mesoderm
with fetal vessels

 

 

Mesoderm

Intervillous space

 

FIG. 3—Secondary chorionic villi surrounded by a thin layer of mesoderm (connective
tissue). The mesoderm is covered by cytotrophoblast and superﬁcially with
syncytiotrophoblast (cells that contact maternal blood). The venous networks are present
within the villi (Gray, 2000 (1918)).

Limiting 01' Stratum spongiosm

boundary
Maternal vessels layerl Plasental septum

 

v ""

Marginal sinus

 

FIG. 4—Tertiary villi and intervillous spaces facilitate placental circulation. Fetal and
maternal blood does not intermingle—the delicate walls of the villi facilitate the
exchange of waste products, and oxygen and nutritive materials. After the exchange,
blood is carried back to the fetus by the umbilical vein (Gray, 2000 (1918)).

 

 

FIG. 5—Photograph of an embryo at 7 weeks contained within the chorionic sac. The
chorionic villi are evenly distributed—covering the chorionic sac (0 ’Rahilly and Muller,
2001).

Forensic STR Analysis

Today, most forensic DNA typing focuses on DNA regions with repeating units
referred to as short tandem repeats, or STRs (Butler, 2001). STRs can be highly variable
due to the repeat unit composition and length of the alleles. The 13 CODIS (Combined
DNA Index System) STR loci (CSFIPO, FGA, THOl, TPOX, vWA, D3Sl358, D5S818,

D78820, D8Sl 179, D13S3l7, D16SS39, D1885], and D2181 1) were selected because of

their high discriminating power (in combination), separate chromosomal locations (to
avoid linkage), result reproducibility and robustness in multiplexing (simultaneous
ampliﬁcation of alleles), low mutation and stutter rates, and allele lengths between 90—
500 base pairs (smaller sizes are best for degraded samples encountered in forensic
testing) (Butler, 2005).

Commercial kits are available that allow multiplex ampliﬁcation of the CODIS
loci. Proﬁler Plus“ and COﬁlerTM (manufactured by Applied Biosystems) are examples
of these, with Proﬁler PlusTM amplifying nine of the loci (D3Sl358, vWA, FGA,

D881 179, D2181 1, D1885], D58818, D13S3l7, and D78820) and COﬁlerTM six
(D3Sl358, D168539, THO], TPOX, CSFIPO, and D78820). An allelic ladder, an
artiﬁcial mixture of all of the common alleles present at each locus, is used as a
“measuring stick” to determine the alleles present within a sample. The combination of
allelic information ﬁ'om the loci comprises a genetic proﬁle. Proﬁles from evidence
samples can be compared to known samples from victims and suspects, and conclusions
regarding the source of the proﬁles can be drawn.

Unfortunately, challenges in interpretation and comparison of forensic STR
results often occur. Microvariants, mutations, DNA degradation, inhibition of
ampliﬁcation, and mixtures of alleles from multiple DNA contributors are commonly
encountered (Butler, 2001). Microvariants are rare alleles differing from a common
allele by one or more nucleotides. These often generate “off-ladder” alleles (OL alleles)
as they are not present in the allelic ladder. Ampliﬁcation must be repeated to conﬁrm
the “OL allele” status of a suspected microvariant. If re-analysis veriﬁes that the allele is

a true microvariant, further comparison with the allelic ladder is necessary. The allele

l3

designation can be interpolated if the microvariant falls within two ladder alleles, or
extrapolated if it falls outside of the allele range. Mutations in STRs usually result in
single base changes or changes in repeat unit. A mutational event can result in a
mismatch between a parent’s DNA proﬁle and a child’s (see the Limitations in
Interpretation of Genetic Testing Results section for further explanation). Degradation
(random breakdown of DNA molecules due to environmental exposure), which is
common in forensic samples, can result in incomplete or failed ampliﬁcation. Larger-
sized loci are more likely to disappear due to degradation, rendering ampliﬁcation of
intact sequences impossible. Partial proﬁles (comprised mainly of results from smaller-
sized loci) are sometimes obtained from degraded samples but often there are no results
observed (false negative). Inhibition (interference of ampliﬁcation), caused by
substances contaminating the DNA, can result in partial or no proﬁle results as well. .A
summary by Wilson (1997) identiﬁed three mechanisms by which inhibitors act—
inactivation of the DNA polymerase, degradation or capture of nucleic acids, and
interference with the lysis of cells during DNA extraction. Numerous substances (e. g.
textile dyes (Shutler et al., 1999), hemoglobin (Akane et. al., 1994; Mercier et al., 1990),
melanin in tissue and hair (Eckhart et al., 2000), polysaccharides and bile salts in feces
(Lantz et al., 1997; Monteiro et al., 1997), humic compounds in soil (Tsai and Olson,
1992), heparin (Beutler et al., 1990), phenol (Katcher and Schwartz, 1994), plant
polysaccharides (Demeke and Adams, 1992), polyamines (spermine and spermadine)
(Ahokas and Erkkela, 1993), urea in urine (Khan et al., 199]; Mahoney et al., 1998),
detergents (i.e. SDS) (Gelfand, 1989), and calcium alginate swab ﬁbers and aluminum

swab shafts (W adowsky et al., 1994)) have been identiﬁed as PCR inhibitors. Careful

14

consideration and selection of sample preparation methods which adequately reduce
inhibitory effects is crucial for optimal ampliﬁcation results (Radstrom etal., 2004).
Finally, mixtures of alleles from multiple contributors can render source attribution
difﬁcult or impossible (see the Limitations in Interpretation of Genetic Testing Results
section for further explanation).
Factors AffectirLLDNA Recovery from Tissue Salees

Romero et a1. (1997) stated that the extraction of DNA from formalin-ﬁxed tissue
embedded in parafﬁn has historically shown little success. Opinions as to the effects of
formalin on tissue have varied over time. Formalin was originally thought to damage
DNA molecules causing strand breakage. It is now understood that the formation of
methyl bridges between amino groups of purine and pyrimidine bases as well as between
the bases and histones, can be facilitated by formalin (Brutlag et al., 1969; Feldman,
1973; Moerkerk et al., 1990; Romero et al., 1997). The formation of these cross-links
can inhibit extraction of DNA ﬁom ﬁxed samples. Karger et a1. (2001) were successful
at obtaining genetic proﬁle information in a limited study of six samples of fetal/maternal
decidua from an abortion. These were obtained from microscope slides prepared from
formalin-ﬁxed parafﬁn-embedded abortion materials. Two of these generated fetal
results.
Organic and Chelex® 100 DNA Extraction of DNA

A popular method for DNA isolation employed in forensic laboratories uses
Chelex® 100. Chelex® 100 is composed of paired inrinodiacetate ions attached to styrene
divinylbenzene copolymers (plastic beads), which chelate (bind) polyvalent metal ions

such as magnesium and iron; these ions can help degrade DNA, or inhibit its subsequent

15

analysis. The Chelex® procedure may be more successful at isolating higher quantities of
DNA for STR-PCR testing (Walsh et al., 1991) than traditional organic extractions
involving phenol/chloroform/isoamyl alcohol (see below). Further, Chelex® 100 resin is
added directly to the sample tube and no DNA transfers are required; this reduces the
chance of sample loss and contamination (Walsh et al., 199]). The sample is boiled in
the presence of the Chelex® beads, which according to Singer-Sarn et a1. (1989) protect
the DNA from degradation. Exposure to boiling (100°C) destroys the cell membranes—
releasing DNA into the solution and denaturing the DNA.

Organic extraction includes the use of proteinase K, a detergent, (e. g., SDS or
Tween 20) and exposure to hot temperatures (e.g., 56 °C) to break down cell
membranes—releasing DNA into solution. The addition of phenol/chloroform/isoamyl
alcohol facilitates a physical separation of the hydrophilic DNA from the hydrophobic
protein materials. (The DNA is more soluble in the aqueous phase of the solution,
whereas, the proteins remain in the organic phase.) Centricon-IOOTM concentrators or
similar devices can be used to purify the DNA, removing small molecules such as
hemoglobin that may inhibit DNA analyses. A risk with this method is the need to
transfer the aqueous DNA containing solution from one tube to another, resulting in
potential sample loss.
Limitations in Interpretation of Genetic Testing Results

According to Karger et a1. (2001), the interpretation of DNA analysis results from
aborted tissues can be challenging; often the results are a mixture of embryo or fetus and
mother. Genetic inheritance is based on the combination of allelic information from both

parents, thus when results are a mixture of embryo/fetus and mother, determination of the

16

embryo/fetal proﬁle can be difﬁcult (Butler, 2001). A known sample from a parent is
crucial to the process of identifying the embryonic or fetal proﬁle. Once the proﬁle is
identiﬁed, suspected father proﬁles can be compared to determine the likelihood of
paunnhy.

Comparison of potential suspect proﬁles to the proﬁle of the embryo/fetus can
also pose challenges, and association of the suspect to the embryo/fetus is never
conclusive. The strongest association possible is that “the suspect cannot be excluded as
a parent of the offspring”. Generation of a paternity likelihood ratio supports the
association—estimating the likelihood that the suspect is a parent of the offspring versus
another random individual. However, results may or may not be easy to interpret.
Sometimes one or more mismatch occurs between the suspect and the offspring, and the
suspect sample is readily excluded as the father. On the other hand, a mismatch between
the offspring and suspect may result from a mutational event and the suspect’s sample
still cannot be excluded. Paternity testing generally allows for one mismatch between the
potential father and offspring due to mutational events (Butler, 2001). Most often this
mutation will result in a difference i one STR repeat unit.

According to Brinkmann et a1. (1998), paternal mutations are more common than
maternal mutations, with a ratio of 17:3, due to the different numbers and types of cell
division. The oogonia divide approximately 22 times before meiosis begins and the
oocyte is formed. The spermatogonia are constantly renewed by mitosis and some
continue to divide through meiosis before becoming sperm cells. The rate of mutation in
older men is even higher than in younger men due to more cell divisions (Brinkmann et

al., 1998).

17

Goal of this Research

The research presented here was designed to evaluate a combination of techniques
for obtaining useful DNA proﬁle information from the analysis of aborted embryonic or
fetal tissue. It was proposed that microscopic identiﬁcation of embryonic structures
(chorionic villi) would reduce the chances of sampling maternal tissue during DNA
testing. The exposure of the tissue to formalin ﬁxative during the parafﬁn embedding
process, although necessary for the preparation of quality microscopic specimens, is
generally not favorable for genetic proﬁling. Chelex® and organic DNA extraction
methods are both designed to generate analyzable DNA, therefore comparison of these
was conducted to determine their relative effectiveness in obtaining DNA, and in
minimizing the negative effects caused by formalin exposure. The success of either
extraction method was determined based on the quantity of DNA recovered and ability to
retrieve full genetic proﬁles from the abortion material. The generation of full proﬁles
would enable comparison to maternal and paternal proﬁles to reveal paternity status.
Ultimately, the goal was to apply the most effective combination of techniques to
forensic casework for the purpose of enabling the most discriminating comparison of
evidence to putative father proﬁles, thus assisting in accurate prosecution of applicable

rape crimes.

18

MATERIALS AND METHODS

Institutionzﬁ Review Board (IRB) Approval

Per UCRIHS (University Committee on Research Involving Human Subjects),
this research project involved only the in vitro use of de-identiﬁed human tissues;
therefore, it did not require IRB approval. A certiﬁcation form was submitted to Dr.
Peter Vasilenko, IRB Chair, Ofﬁce of Research Ethics and Standards, Michigan State
University and approved under certiﬁcation #CT06-002.
Sample Collection and Preparation

Prior to this experiment, early-terrn abortions were conducted at undisclosed
facilities in Michigan and the aborted tissue was sent to Dr. Joyce deJong, Medical
Director of Forensic Pathology at Sparrow Hospital, Lansing, MI. The resultant decidua
and embryonic/fetal material from each abortion were examined for identiﬁable fetal
parts. None were located. The tissue from each abortion was sliced into segments less
than one centimeter in any dimension and placed into separate standard tissue embedding
cassettes. Three sets of cassettes (Test Set 1 (TSl-l—IO), Test Set 2 (T82-1—20), and Test
Set 3 (TS3-A—F, I—K, M—O, Q—W)) were prepared and labeled accordingly. Each set of
cassettes was transferred to the Sparrow Hospital Department of Histology for
processing.
Fixation

The histologist placed the cassettes containing tissue into a basket and then into a

chamber containing 10% formalin at neutral pH. The tissue was ﬁxed in formalin for a

19

period of approximately 10—12 hours (recommended by Greer et al. (1991) and Rogers et
al. (1990)).
Tissue Processing

A tissue processor was used to gradually dehydrate the formalin-ﬁxed tissue. The
tissue was washed with 10% formalin and then passed through increasing strengths of
ethyl alcohol (70%, 80%, 95%, and 100%). Following dehydration, xylene was used to
clear the tissue of the ethyl alcohol. The cassettes were placed into metal cassette holders
and passed through several changes of melted parafﬁn until completely embedded (in
blocks of parafﬁn within the cassettes).
Sectioning

The paraffm embedded tissue was sectioned into 4.0 um slices using a microtome.
The sections were ﬂoated on a warm water bath to remove wrinkles and folds. They
were mounted on slides by placing the slide underneath the section and lifting it out of
the water. A ﬁxative on the slides enabled the tissue to adhere.
Staining

Parafﬁn was removed from the sectioned tissue on the slides with xylene,
followed by 100% ethyl alcohol and water. The tissue was exposed to the stain
hematoxylin, followed by 80% ethyl alcohol, 100% ethyl alcohol, the stain eosin, 100%
ethyl alcohol again and ﬁnally xylene. The slides were placed into a processor for
permanent placement of cover slips. Slides were scanned for quality control and

transferred back to the pathologist.

20

Microscopic Examination

Microscopic evaluation of the slides identiﬁed cassettes containing chorionic villi.
These cassettes were noted. All of the cassettes (and slides) were transferred to the
Michigan State Police Biology Unit for DNA analysis.
Preparation of Parafﬁn-Embedded Embryonic or Fetal Tissue and Maternal Decidua for

DNA Extgction

 

Sample Preparation

F iﬁy microscope slides corresponding to speciﬁc parafﬁn blocks (Fig. 6) (sets
identiﬁed as T81, T82, and T83) were visually analyzed and locations containing
chorionic villi were identiﬁed (see Appendix C for complete protocol). Two 2—4 mm3
tissue segments were cut ﬁom each parafﬁn block (areas corresponding to chorionic villi
identiﬁed on the slide) using a sterile razor blade, and were placed into separate labeled
microcentrifuge tubes for parafﬁn removal followed by digestion and Chelex® or organic
extraction. A blank tube (no tissue segment) was prepared for each extraction method,
and was carried through the entire extraction process with the purpose of identifying
contamination, if present, in the reagents used. The tubes containing tissue segments and
the reagent blank tubes were collectively referred to as ‘samples’ from this point forward

in the experiment.

21

 

FIG. 6—Photograph of the paraﬂin block containing sample TS1—4A. The slide was
orientated over the paraffin block consistent with the corresponding tissue on the slide
and in the block
Deparaﬁ‘inisation

A xylene/ethanol deparafﬁnisation method (Coombs et al., 1999; Goelz et al.,
1985) was utilized on both sets of ﬁfty samples. A 1 mL aliquot of xylene was added to
each of the samples to remove the paraffin wax (see Appendix C for complete protocol);
the samples were then incubated for 30 minutes at room temperature, and then
centriﬁrged for 2—5 rrrinutes at 15,300 relative centrifugal force (RCF). The liquid
portion was discarded and the process repeated. A 1 mL aliquot of ethanol was added to
each of the samples to remove the remaining xylene item the tissue. The samples were
centrifuged at 15,300 RCF for 2—5 minutes. The liquid portion was discarded and the

process repeated. The samples were dried in a Hetovac vacuum apparatus at 15—20 in.

Hg for 10-20 minutes.

22

Digestion of T issue, Purification of DNA and Concentration of DNA

Both sets of 50 samples were digested according to Kawaski (1990), Sepp et al.
(1994), and Shimizu and Burns (1995). A 200 uL aliquot of ﬁltered tissue lysis buffer
(189 uL TE4 (10 mM Trizrna base, pH 7.5; 0.5 mM EDTA, pH 7.5), 10 uL 0.5% Tween
20, and 10 uL proteinase K (20 mg/mL)) was added to each of the samples prior to
overnight incubation (12—1 8 hours) at 37°C (see Appendix C for complete protocol).
The samples were centrifuged for 5 minutes at 15,300 RCF. Centricon-IOOW'
concentrators were assembled according to the manufacturer’s instructions for each
sample. The sample reservoirs (containing the ﬁlter unit) with attached rententate vials
were ﬁtted to ﬁltrate vials. The liquid portion of each digested sample (approximately
200 uL) was placed into a separate Centricon-l 00““ concentrator sample reservoir (the
tissue was discarded), and centrifuged for 30-60 minutes at 2000 RCF. The ﬁltrate from
each was discarded. A 2 mL aliquot of TE“1 was added to each sample, and the samples
were centrifuged for 30-60 nrinutes at 2000 RCF. The ﬁltrate was discarded and the
process repeated. Following the second wash, the ﬁltrate vials were removed ﬁom the
concentrators and discarded. The sample reservoirs with attached retentate vials were
inverted and centrifuged at 1000 RCF for 3 minutes. The concentrated DNA (rententate)
was captured in the retentate vials and transferred to clean, labeled, microcentrifuge tubes
was used for Chelex® 100 extraction and the other for organic extraction.
DNA Extraction of Embryonic or Fetal—Tissue and Maternal Decidua

One sample from each of the 50 cassettes was extracted with Chelex® (Walsh et
al., 1991) and one was extracted organically (phenol/chloroform/isoamyl alcohol)

(Sambrook et al., 1989).

23

Chelex® Extraction

A solution of 5% Chelex® 100 was prepared by adding 2.5 g of Chelex® resin
beads to 50 mL of sterile water and mixing until evenly distributed (see Appendix D for
complete protocol). The pH was veriﬁed at 9.0 using a Coming 220 pH Meter and buffer
solutions of pH 7.0 and pH 10.0. A 20 uL aliquot of the Chelex® 100 solution was added
to each tube. The samples were vortexed brieﬂy and incubated at 56°C for 30 minutes.
The samples were then vortexed at high-speed for 5—10 seconds and placed into a boiling
water bath for 8 minutes. These were vortexed again at high-speed for 5—1 0 seconds and
centriﬁrged for 3 minutes at 15,300 RCF.
Organic (phenol/chloroform/isoamyl alcohol) Extraction

A 200 uL aliquot of phenol/chloroform/isoamyl alcohol 25:24:] was added to
each sample (see Appendix E for complete protocol). The samples were vortexed until a
milky emulsion was achieved (5—10 seconds) and then centrifuged at 15,300 RCF for 5
minutes. After centrifugation the components were separated into a lower organic
solution, an interface layer of protein and cellular material, and an upper aqueous portion.
The aqueous portions were transferred to clean Centricon-IOOTM concentrators and
centrifuged for 30-60 minutes at 2000 RCF (the ﬁltrate was discarded). A 2 mL aliquot
of TE'4 was added to each sample, and the samples were centrifuged for 30-60 minutes at
2000 RCF. The ﬁltrate from each was discarded and the process repeated. Following the
second wash, the ﬁltrate vials were removed from the concentrators and were discarded.
The sample reservoirs with attached retentate vials were inverted and centrifuged at 1000
RCF for 3 minutes. The concentrated DNA (rententate) was captured in the retentate

vials and was transferred to clean, labeled, microcentrifuge tubes.

24

Quantiﬁcation of DNA

The Chelex® and organically extracted samples and kit standards (known DNA
quantities of 10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.625 ng, 0.3125 ng, and 0.15625 ng) were
quantiﬁed using a QuantiblotTM kit (manufactured by Applied Biosystems) according to
the manufacturer’s instructions. The chemiluminescent method of detection was used.
Membranes were placed on Kodak® X-Omat L8 ﬁlm and exposed overnight (24 hours).
The ﬁlms were processed using a medical ﬁlm processor and compatible chemistry. An
estimate of DNA quantity was made in relation to the standards. If no DNA was
detected, those samples were concentrated to 10 uL using Microcon-100TM Micro-
concentrators according to manufacturer’s instructions.
DNA Ampliﬁcation

A PCR master mix was prepared with primers, reaction mix, and AmpliTaq
GoldTM DNA polymerase from AmpFlSTR Proﬁler PlusTM and AmpFlSTR CoFilerTM
kits with one minor modiﬁcation to the manufacturer’s instructions (total reaction volume
per sample was lowered to 25 uL). Samples with no detectable DNA based on
QuantiblotTM results were ampliﬁed with the Proﬁler PlusTM kit only using any/ all
available DNA. Dilutions or concentrations of the samples (whichever was appropriate
given the estimated quantity of DNA detected) and the positive control samples were
prepared; targeting 1.0 ng of DNA per 10 uL of sample based on the QuantiblotTM
estimates. Fifteen microliters of master mix (10.5 uL of PCR Reaction mix, 5.5 pL of the
Proﬁler PlusTM or COﬁlerTM kit primers, and 0.5 uL of AmpliTaq Gold”) was combined
with 10 uL of each sample and control. The negative controls were prepared with 10 uL

of sterile water replacing the DNA. All were ampliﬁed using ABI 9700 thermocyclers

25

with an initial incubation at 95°C for 11 minutes to activate the AmpliTaq GoldTM
enzyme. Denaturing was conducted at 94°C for 1 minute, primer annealing at 59°C for 1
minute, and extension at 72°C for 1 minute. This sequence was repeated for a total of 28
cycles. The ﬁnal extension was conducted at 60°C for 45 minutes, and the plates were
held at 25°C until removal from the thermocycler.
Sample Preparation for Electrophoresis and GeneScan Analysis
One microliter of each ampliﬁed sample, control product, or allelic ladder
standard (Proﬁler PlusTM and COﬁlerTM) was added to 24 uL of deionized formamide
(Bio-Rad Laboratories) and 1 uL of GS-500TM ROX internal size standard. The samples,
controls and ladders were denatured at 95°C for 3—5 minutes and snap-cooled on ice for a
minimum of 3 minutes. An ABI PrismTM 310 Genetic Analyzer and ABI PrismTM 310
Collection Software were used according to manufacturer’s instructions to obtain raw
data of genetic proﬁles. These were analyzed with GeneScan® Analysis Software, while
Genotyper® software was used to obtain ﬁnal allele designations at each locus.
Data Interpretation
Determination of Full vs. Partial Single-Source Proﬁles
The guidelines for interpretation of acceptable single-source genetic proﬁle
results were:
1. Each allele peak must fall within a minimum threshold of 150 relative
ﬂuorescent units (RFUs) and a maximum threshold of 4500 RFUs
(relative ﬂuorescent units), with the exception of the arnelogenin locus
which has a maximum threshold of 7500 RFUs to qualify for

interpretation.

26

2. Allelic balance for heterozygosity must equal or exceed 70 percent.
Single-source proﬁles were considered full (complete) if alleles at each of the loci
(Proﬁler Plus—D3Sl358, vWA, FGA, D881179, D218] 1, D1885], D58818, D1383 1 7,
and D78820; COﬁler—D3Sl358, THO], TPOX, CSFIPO, D78820, and D168539) fell
within the 150—4500 RF Us range. The sample was also considered to have generated a
full proﬁle if enhancement utilizing a 3 pL input of ampliﬁed DNA or a decreased
injection time of 1—4 seconds was expected to place all alleles within the range (deﬁned
above). Compilation of results from multiple electropherograms from the same sample
was acceptable as well. Samples which generated interpretable results at the amelogenin
locus and at least one allele were considered partial proﬁles. A sample was also
considered a partial proﬁle if enhancement utilizing a 3 uL preparation of ampliﬁed DNA
was expected to place the allele(s) at the amelogenin locus and at least one additional
allele above the minimum threshold of 150 RFUs.

Determination of Full vs. Partial Mixture Proﬁles

A mixture proﬁle (DNA types detected ﬁom more than one donor) was
considered full if alleles from at least one of the two contributors fell within the
established range of RFUs. Samples which did not meet these criteria were considered
ﬁrll proﬁles if enhancement utilizing a 3 uL preparation of ampliﬁed DNA or a decreased
injection time of 1—4 seconds was expected to place all alleles from at least one
contributor within the interpretable range of RFUs. Successful interpretation of loci over
multiple electropherograms from the same sample was also acceptable for mixture
samples. Partial proﬁles included interpretable amelogenin, in addition to, a minimum of

one allele at one locus. A mixture sample was also considered a partial proﬁle if

27

enhancement utilizing a 3 uL preparation of ampliﬁed DNA was expected to place the
allele(s) at the amelogenin locus and at least one additional allele above the minimum
threshold of 150 RFUs.
Mixture Allele Relationships Representative of the Mother and a Full Oﬂfspring

Proﬁles which exhibited a mixture of maternal and fetal alleles were recognized
by the presence of several different allele conﬁgurations. If three alleles were present at
one locus, one of them should be shared by the mother and fetus. This allele would be
consistent with the proportion of the contribution from the mother and from the fetus (the
other two alleles) combined. In a two—allele result the mother and fetus must be
heterozygous and share the same two alleles, or one must be homozygous and the other
must be heterozygous. In the former circumstance, the alleles would be equal in
contribution; in the latter circumstance, the shared allele of the homozygous contributor
and the heterozygous contributor should be three times as large as the remaining allele
contribution (assuming a 50/50 mixture of contributors). A single allele result would
indicate that the embryo/ fetus and mother were both homozygous sharing the same allele.
If the maternal proﬁle is known, which it was not in this study, it is generally simple to
discern the fetal proﬁle. Additionally, if both the maternal and putative father’s proﬁles
are known (also not known in this research), this task becomes even easier with fewer
assumptions.
Sample Concordance

Samples were compared between ampliﬁcation sets at overlapping loci D381 358,
D78820 and amelogenin, as well as between extraction sets at all loci to determine if the

results were concordant. Allele designations and ratios were expected to be identical for

28

IF:

the overlapping loci of Proﬁler PlusTM- and COﬁlerTM- ampliﬁed samples with the
exception of the presence of additional minor contributor alleles in COﬁlerTM due to
greater observed sensitivity at the Lansing Laboratory. It was anticipated that the
Chelex®-extracted and organically-extracted samples could vary in the ratio of allelic
contribution between mother and offspring as a result of adjacent sampling. Both sets of
samples needed to exhibit at least one identical allele designation at each locus to indicate
concordance.

While comparing samples for concordance, relationships among samples were
identiﬁed. Allele designations within each extraction set and between extraction sets
were compared and samples that shared one or more alleles at each locus were considered
to be associated. Some samples were compared using only one ampliﬁcation system or
one extraction method due to limited proﬁle information.

Statistical Analyses

DNA yields resulting from the organic and Chelex® extraction methods were
compared. A mean of the DNA yield results was calculated for each of the methods.
This value was used to conduct a two-tailed t test using the separate variance estimate
(Bachman and Paternoster, 1997), indicating whether a signiﬁcant difference in recovery

of DNA existed between extraction methods. The following formula was used:

t 2

ob! \/’ s] + $2
("r-1) ("z—1)

x1 = sample mean from ﬁrst sample set

x; = sample mean from second sample set
s. = sample standard deviation of ﬁrst set

32 = sample standard deviation of second set
n. = sample size ﬁrst set

112 = sample size second set

 

29

The following formula was used to calculate the degrees of freedom (df):

_ 2 2 -
s1 + s2
nl-l nz—l

(If; -2

sf 2___1__+ s22 2 1
Lnl-l nl+l nz—l n2+l a

$1 = sample standard deviation of ﬁrst set

32 = sample standard deviation of second set
n. = sample size ﬁrst set

112 = sample size second set

 

 

 

 

 

 

 

The result was rounded to the nearest integer to obtain the approximate degrees of
freedom. The null hypothesis to be tested was that no signiﬁcant difference in DNA
recovery existed between the two extraction processes.

A 2 test (Bachman and Paternoster, 1997) based on a proportion calculation of
extraction attempts and the actual recovery of a full genetic proﬁle was used to determine
whether or not a signiﬁcant difference existed between methods in obtaining full genetic

proﬁles. The following formula was used:

 

Z =[(PITP2)-(Pi—P2)]
ab! 0'
P142

Apl = the sample proportion for the ﬁrst sample

Apz = the sample proportion for the second sample

p1 = the ﬁrst population proportion

p2 = the second population proportion

op]_p2 = the standard error of the difference between proportions

The pooled standard error was calculated with the following formula:

nl+n2

 

nrnz

30

The result was then used to calculate the 20b, value. A 95% conﬁdence interval was
selected with a critical region 2 score of i 1.96. The null hypothesis to be tested was that
no signiﬁcant difference existed between extraction processes in obtaining full genetic

proﬁles.

31

 

Jm_1

RESULTS

Parafﬁn Extraction

Upon sampling, the embryonic/fetal tissue was ﬁrm in texture and solid in
appearance due to the support of the parafﬁn wax. Xylene exposure effectively
solubilized the parafﬁn wax—releasing it from the tissue. At this point, the tissue was
pliable and soft with a sponge-like appearance. The addition of alcohol cleared the
xylene from the tissue. After removal of the ﬁnal alcohol solution, the xylene odor was
not detectable. Traces of alcohol were removed from the tissue through evaporation.
DNA Quantiﬁcation

Human DNA was detected in all of the 50 Chelex®-extracted samples utilizing the
QuantiblotTM kit (Tables 1—3). The quantity of DNA recovered ranged ﬁ'om 25 ng to
1500 ng. DNA was detected in 36 of the 50 organically-extracted samples (72%) (Tables
4—6). Samples exhibited a range of DNA recovery from 0 ng to approximately 400 ng.
Figures 7—9 illustrate the comparison of DNA quantity recovered from each Chelex® and
organically extracted sample. Calculation of the percent difference in recovery revealed
that 73.1% more DNA was acquired using the Chelex® method than with the organic

method.

32

TABLE l—QuantiblotTM results of human DNA recovered ﬁom Chelex® extraction of
Test Set 1 samples

. Sampleld# p.L* ng/HLT ngtot'alzﬂ

T8] 1 100 2.0 200
T81 3 100 10.0 1000

 

TS1 4B 100 10.0 i000

 

TSl 6 100 5.0 500

 

T8] 8 125

 

 

T81 10 100 15.0 1500

*Volume of supernatant recovered from the Chelex® extraction method
*Quantity of DNA contained within one microliter of DNA sample
zTotal quantity of DNA contained within the DNA sample

33

TABLE 2—QuantiblotTM results of human DNA recovered from Chelex® extraction of
Test Set 2 samples

Sample Id#
T82 1
T82 2
T82 3
T82 4
T82 5

2 T82 6
T82 7
T82 8 '
TS2 9

' TS2 10
T82 11
T82 12
T82 13
T82 14
T82 15
TS2 16
T82 17
TS2 18
T82 19
T82 20

“13"
100
100
100
100
100

. 100

100
100
100

. 100

100
100
100
150
100
100
150
100
100
350

ng/pLT
3.0
2.5
12.5
0.625
1.5
12.5 *
0.625
1.5
2.5
0.5
2.5
2.5
10.0
0.625
1.0
15.0
1.0
2.5
2.0
2.0

*Volume of supernatant recovered from the Chelex® extraction method
lQuantity of DNA contained within one microliter of DNA sample
*Total quantity of DNA contained within the DNA sample

ng total1
300
250
1250
62.5
150
1250
62.5
I 150
250
'50 .
250
250
1000
93.8
100
1500
150
250
200
700

TABLE 3-——QuantiblotTM results of human DNA recovered from Chelex® extraction of

Test Set 3 samples
sample Id# nu ‘ ng/nU ng total: '
T33 A 100 2.0 200
TS3 B 175 2.0 350
T33 c 100 3.5 350
TS3 D 150 ‘ 2.5 _ 375
TS3 E 100 5.0 500
TS3 F 100 2.5 250
TS3 I 175 3.5 612.5
TS3 J 100 2.0 200
T83 K 100 3.5 350
TS3 M 150 2.5 375
T33 N 100 7.5 750
TS3 o 100 3.5 350
T33 Q 100 0.5 50
T33 R 100 2.5 250
T33 3 100 2.5 250
T33 T 100 3.5 350
T33 U 100 2.0 200
T33 V 150 3.5 525 _
TS3 w 200 2.0 400

*Volume of supernatant recovered from the Chelex® extraction method
TQuantity of DNA contained within one microliter of DNA sample
ITotal quantity of DNA contained within the DNA sample

35

 

TABLE 4—QuantiblotTM results of human DNA recovered from organic extraction of
Test Set 1 samples

Sample Id# 4 uL“ I ' ng/ 11Lf . i ng totalI
T31 1 100 0.3125 31.25
, T311 2 150 ‘ 0.3125 46.88
T31 3 125 0.3125 39.06
TSl.4Af 100‘ 1.75 175
T81 43 75 0 0
TSl 5 100 0.3125 31.25
T316 100 0 0
T31 7 125 0.3125 16.56
T318 100 o 0
T819 125 0.5 62.5
T31 10 125 0.15625 19.531

*Volume of supernatant recovered from the organic extraction method
TQuantity of DNA contained within one microliter of DNA sample
ITotal quantity of DNA contained within the DNA sample

36

 

TABLE S—QuantiblotTM results of human DNA recovered from organic extraction of

Test Set 2 samples
Sample Id# pL“ ng/uLl ng totali

T82 1 175 0 0
T82 2 100 O O
T82 3 100 1.75 175
T82 4 100 0 0
TS2 5 100 0.3125 31.25
T82 6 100 0.15625 15.625
T82 7 100 0 0
T82 8 100 0 0
T82 9 100 0.3125 31.25
T32 10 100 0 0
TS2 11 100 0 0
T82 12 100 , 7 0 0
T82 13 100 0 0
T82 14 150 1.25 , p . '- 125
T82 15 100 0 0
T82 16 150 0.3125 46.88
TS2 17 75 1.25 93.8
TS2 18 75 1.75 131.3
T82 19 50 1.25 62.5
T82 20 100 0 0

*Volume of supernatant recovered from the organic extraction method
“Quantity of DNA contained within one microliter of DNA sample
iTotal quantity of DNA contained within the DNA sample

37

 

 

TABLE 6—QuantiblotTM results of human DNA recovered ﬁom organic extraction of

Test Set 3 samples
Sample Id# uL“ U ng/uLT ng total‘t
T33 A 175 0.3125 54.69
T33 B ' 300 0.3125 93.757
T33 c 200 0.5 100
T33 D ‘ 150 0.5 75
TS3 E 300 0.3125 93.75
T33 F 125 0.5 62.5
T33 1 150 0.625 93.8
T33 J 75 1.75 131 f
TS3 K 175 0.625 109
T33 M 75 5 375
TS3 N 75 1.75 131
TS3 o 75 2.5 187.5
T33 Q 75 1.25 93.8
TS3 R 75 2 150
T33 s 75 2 150
T33 T 75 1.25 93.8
T33 U 75 1.5 112.5
TS3 v 75 2.5 187.5
T33 w 75 1.5 112.5

*Volume of supernatant recovered from the organic extraction method
TQuantity of DNA contained within one microliter of DNA sample
ITotal quantity of DNA contained within the DNA sample

38

 

1 600

 

1 400

1 200

800

600 A

400

200 ‘ 1
3

T81 1 1’81 2 T81 T31 4A T81 43 T81 5 TS1 6 TS1 7 T51 8 TS1 9 T81 10

 

 

 

 

 

total ng recovered

 

 

 

 

 

 

 
 

 

 

 

    

 

 

 

Sample

FIG. 7—Comparison of DNA recovery from T S-I Chelex®- and organically-extracted
samples. Odd columns (black) represent Chelex®-extracted samples. Even columns
(gray) represent organically-extracted samples. Sample quantities were obtained using
the QuantiblotTM kit procedure and were measured in ng.

39

 

1600

1400

 

 

 

 

total ng recovered
§

600

 

400

200 ~
4
w ..
‘2': -'
a. ‘1 r?
0 ‘ h. Sn .

TS2 TS2 TS2 T32 T82 T82 TS2 T82 TS2 T82 T82 T82 T82 T82 T82 T82 T82 T82 T82 TS2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

 

 

 

1‘14“; a”:

. +1473 2

.ILA-ftrlo

 

run
‘I

 

 

Sample

FIG. 8—Comparison of DNA recovery from T S-2 Chelex®- and organically-extracted
samples. Odd columns (black) represent Chelex®-extracted samples. Even columns
(gray) represent organically-extracted samples. Sample quantities were obtained using
the QuantiblotTM kit procedure and were measured in ng.

40

 

700

 

600

 

500

 

400

 

total ng recovered

300-

 

200‘

r“, 1V A

'a« 1311 ~:,_‘m
.r , 1 ..
.‘m‘! I‘g‘rLex‘. .-'

100-

L

f. ("b

l

_§hﬁ
l- ‘7 1.’

 

 

 

 

Vii-33$" ..

T83 T83 T83 T83 T83 T83 T83 T83 T83 T83 T83 T83 T83 T83 T83 T83 T83 T83 T83
A B C D E F l J K M N O Q R S T U V W

FIG. 9—Comparison of DNA recovery from T S-3 Chelex®- and organically-extracted
samples. Odd columns (black) represent Chelex®-extracted samples. Even columns
(gray) represent organically-extracted samples. Sample quantities were obtained using
the QuantiblotTM kit procedure and were measured in ng.

41

Ampliﬁcation and Electrophoresis of Puriﬁed DNA

Results for all of the thirteen CODIS core loci and amelogenin were obtained
from 48 of the Chelex®-extracted samples (96%) ampliﬁed with the Proﬁler PlusTM
Ampliﬁcation kit, while 37 of the organically-extracted samples (74%) generated full
proﬁles (Fig. 10; Tables 7—9). Results using COﬁlerTM included 48 of the samples
extracted with Chelex® (96%) and 15 of the organically-extracted samples (30%). All

control samples performed as expected.

42

 

 

# of Proﬁles

 

 

 

 

 

Proﬁler Plus COﬁlcr
Extraction Method

FIG. lO—Number of full profiles generated for Chelex01 extraction (black—1‘" and 3rd
columns) and organic extraction (gray—2"“! and 4’hcolumns). Full profiles were
generated for 48 samples (96%) extracted with Chelex® and ampliﬁed with the Profiler
PlusTM amplification kit (black—1“ column). Full proﬁles were generated for 3 7 samples
( 74%) extracted organically and amplified with the Profiler PlusTM amplification kit
(white—2'” column). Full proﬁles were generated for 48 samples (96%) extracted with
Chelex‘R and amplified with the COﬁlerTM ampliﬁcation kit (black—3rd column). Full
proﬁles were generated for 15 samples (30%) extracted organically and amplified with
the COﬁlerTM amplification kit (white—«4“ column).

43

TABLE 7—Full genetic proﬁles generated for Test Set 1 samples

Chelex® ' Chelex® ' Organic A +" Organic

Sample Id# Proﬁler COﬁlerTM‘i Proﬁler PlusTM+ COﬁlerTM§
_ Plusm.“ . . , . .

T311 1—E> 1—E> 1—E> l—E>

T312 1'—1~:> 1—13> 0—P(9) 0-P(3)
T313 1 1 0—P(10) 0—P(7)

,. TS14A. 1 1 — 13> i0—P (8) p 01- P (5)
TS14B 1 1 1—E>&E< l—E<
T315 1 1 1 4 E> 1 — 13>
T316 1—E> 1 0—P(5) O—NR
T317 f 1 1—E< 1 1—E>&E<
T318 1—E> l—E< l—E> 1—B>&E<
T31 9 1 ' 1 1 —E> 04) (6)
T3110 1 1 l—E> 0—P(5)

*Chelex®-extracted and Proﬁler PlusTM-ampliﬁed samples

IChelex®-extracted and COﬁlerTM-ampliﬁed samples

IOrganically-extracted and Proﬁler PlusTM-ampliﬁed samples

§Organically-extracted and COﬁlerTM-ampliﬁed samples

1, a full genetic proﬁle with interpretable alleles at all 10 loci for Proﬁler PlusTM and 7
loci for COﬁlerTM was obtained; 0, a full genetic proﬁle with interpretable alleles was
not obtained; P, partial proﬁle detected & ( ), complete loci generated; NR, ampliﬁcation
not conducted; E>, enhancement required 3 11L preparation; E<, enhancement required 1—
4 second injection

TABLE 8—Full genetic proﬁles generated for Test Set 2 samples

Chelex® Chelex® Organic Organic
Sample Id# Proﬁler COﬁlerTM‘t Proﬁler PlusTM“ COﬁlerTM§
Plusm“

T82 l l 1 1 — E> 0 — NR
T822 l-E< 1—E< 0—P(8) O—NR
T82 3 l 1 - E< 1 1 — E<
T824 1—E> 1—E> 1—E> 0-NR
T825 l—E> 0—P(5) 1—E< 1—E<

' T82 6 1 —- E< 1 — E< 1 1
T827 0—P(2),E> 1—E> 1—E> 0—NR
T82 8 1 1 l 0 — NR
T82 9 1 l 0 — ND 0 — ND
'T8210 1 l l—E< 0—NR
T82 11 1 1 1 0 — NR
T8212 1 1 l E< _0-—NR
T8213 1—E< 1—E< l- E> O—NR
T82 14 1 — E> l 0— ND 0 — NR
T82 15 1 1 1 0 — NR
T8216 l—E< l 0—P(2) 0—P(6)
T8217 0—P(8) l 0—P(9) 0—NR
T8218 1 l 0—P(3),E> O-NR

T82 19 1 1 1 — E> l
T8220 l—E< l 0-ND O—NR

*Chelex®-extracted and Proﬁler PlusTM-ampliﬁed samples

Chelex® -extracted and COﬁlerTM-ampliﬁed samples

*Organically-extracted and Proﬁler PlusTM-ampliﬁed samples

§Organically-extracted and COﬁlerTM-ampliﬁed samples

1, a full genetic proﬁle with interpretable alleles at all 10 loci for Proﬁler PlusTM and 7
loci for COﬁlerTM was obtained; 0, a ﬁll] genetic proﬁle with interpretable alleles was
not obtained; P, partial proﬁle detected & ( ), complete loci generated; NR, ampliﬁcation
not conducted; E>, enhancement required 3 11L preparation; E<, enhancement required 1—
4 second injection

45

TABLE 9—Full genetic proﬁles generated for Test Set 3 samples

Chelex® Chelex® ' Organic 1 ‘ Organic
Sample Id# Proﬁler COﬁlerTM)‘ Proﬁler Plusmi COﬁlerTm
PlusTM“
T33A 1—E< l—E< 0—P(5) O—NR
T3313 1-E< 1 lV—E> ' O—NR
TS3C 1 1 0—P(9) O—NR
"T3313 1—E> 1 1—E<’ O—NR
TS3E 1—E< 1 1—1~:> O—NR
T33 F A 1 1 — E< 1 — 13> o — NR
T331 1—E< 1 l—E< 0—NR
T331 1 1—E< l—E< l—E<,
TS3K 1—E> 1 1—E> O—NR
T83 M, ,1-;E< 1—E<, 1—,E< 1_-E<
TS3N l—E< 1 1—E< l—E<
”T830 1eE< 1——E< 1-E< O—NR
TS3Q 1—E>&E< l—E< 1-E< O-NR
TS3R 1 1—E< 1—E< o—NR
T333 1 1—E< 1—E< 0—P(5)
TS3T' 1—E< 1-E< 1-‘E< O—NR
TS3U 1 0—P(5) l—E> 1—E<
T33 v 1 — E< 1 1 1
T33 w 1 1 1 1 — E<

*Chelex®-extracted and Proﬁler PlusTM-ampliﬁed samples

TChelex®-extracted and COﬁlerTM-ampliﬁed samples

2tOrganically-extracted and Proﬁler PlusTM-ampliﬁed samples

§Organically-extracted and COﬁlerTM-ampliﬁed samples

1, a full genetic proﬁle with interpretable alleles at all 10 loci for Proﬁler PlusTM and 7
loci for COﬁlerTM was obtained; 0, a full genetic proﬁle with interpretable alleles was
not obtained; P, partial proﬁle detected & ( ), complete loci generated; NR, ampliﬁcation
not conducted; E>, enhancement required 3 11L preparation; E<, enhancement required 1—
4 second injection

T81—Proﬁle Results

 

All of the Chelex®-extracted T81 samples ampliﬁed using the Proﬁler PlusTM and
COﬁlerTM kits and each exhibited full proﬁle results. Likewise, all organically-extracted

T81 samples ampliﬁed using the Proﬁler PlusTM kit, however only seven generated full

46

proﬁles while four were partial. Ten of the organically-extracted TSl samples ampliﬁed
using the COﬁlerTM kit; ﬁve resulted in full proﬁles and ﬁve in partial proﬁles.
T82—Proﬁle Results

The 20 T82 samples extracted with Chelex® ampliﬁed using the Proﬁler PlusTM
and COﬁlerTM kits. Eighteen samples ampliﬁed using Proﬁler PlusTM and nineteen
samples ampliﬁed using COﬁlerTM produced full proﬁles, while two and one produced
partial proﬁles respectively. The partial proﬁles originated from different samples (Table
8).

Of the 20 organically-extracted samples ampliﬁed using the Proﬁler PlusTM kit—
13 produced full proﬁles, four were partial, and three showed no results (reasons
undetermined). Only six organically-extracted samples were ampliﬁed using the
COﬁlerTM kit—four generated full proﬁles, one was partial and no results were detected
for one. Ampliﬁcation of the other 14 samples was not conducted due to insufﬁcient
quantities of DNA. These samples were concentrated and consumed for Proﬁler PlusTM
ampliﬁcation due to the limited quantity of DNA present.
T83—Proﬁle Results

All 19 of the Chelex®-extracted T83 samples ampliﬁed using the Proﬁler PlusTM
and COﬁlerTM kits. Nineteen samples ampliﬁed with Proﬁler PlusTM produced full
proﬁles. Eighteen samples ampliﬁed using COﬁlerTM generated ﬁlll proﬁles and one
produced a partial proﬁle.

Nineteen of the organically-extracted T83 samples ampliﬁed using the Proﬁler
PlusTM kit and full proﬁles were generated for seventeen of them; two produced partial

proﬁles. Of seven samples ampliﬁed using the COﬁlerTM kit, six resulted in full proﬁles

47

and one a partial proﬁle. Due to the limited quantity of DNA present, the other 12
samples were not ampliﬁed, and instead were concentrated and consumed for Proﬁler
PlusTM ampliﬁcation.
Genetic Proﬁle Composition

Electropherograms of Chelex®- and organically-extracted samples were evaluated
and compared. Based on the results, six categories were appropriate for explanation of
the genetic information obtained—single-source female (Fig. 11), single-source female
with additional activity (Fig. 12), single-source male (Fig. 13), single-source male with
additional activity (Fig. 14), mixture of females (Fig. 15), and mixture of a female and a
male (Fig. 16). Samples were categorized by allele presence and balance at the
amelogenin locus, by the number of alleles present at the other loci and the balance
between those alleles. Additionally, the presence of any allele at any locus which did not
meet the minimum threshold for reporting (150 RFUs) and was not an artifact (pull-up,
ﬂuorescent spike, or noisy baseline) was termed ‘activity’ and suggested an additional
DNA contributor. Results which could be attributed to an embryo/fetus were observed in
the following sample categories: single-source male, single-source male or female with
additional activity, mixture of females, and mixture of a female and a male. Source
attribution (embryo/fetus or mother) of the single-source female proﬁles and the single-
source female with additional activity proﬁles was not discemable without comparison to

known samples ﬁ'om the mothers.

48

    

 

 

 

 

 

 

 

 

 

 

50 100 150 200 250 300 350 400
A1 -PPTS1-1 2 Blue PPT81-1
vWA PC A 800
D31358 500
00
200
lst box = allele call
2nd box = base pair size
3rd box = RFU signal strength
(peak height)
A1 -PPTSl-1 2 Green PPTSl-l
4000
amelogenin 088] 179 D2181] D1885 l 3000
2000
1000
1 A 1 7
IE
299.64
260'
18
308.1 1
326
A1-PPTS1-1 2 Yellow PPTSl -1
D13S3l7 D78820 900
D58818 600
00
2-1-2----“ -__- ._ 222.2 -

 

 

 

 

 

 

FIG. ll—Sample T S] -I (extracted with Chelex® and ampliﬁed with the Proﬁler PlusTM
ampliﬁcation kit) exhibited a full single-source female proﬁle. Alleles at the D1353] 7
locus did not ﬁt the 70% rulefor interpretation. With 3 uL enhancement, this proﬁle was
expected to be interpretable at all loci shown and the imbalance at D1353] 7 may be

resolved.

49

 

 

 

I I I I I I I I l I I I I I ' I I I I
50 100 150 200 250 300 350 400
A7-PF’T 81 -7 8 Blue PPTS1-7
vWA PO A 3000
D38 1358 2000
I 1000
AA- - l A. _
lst box = allele call
2nd box = base pair size

[131.74] Bum
3rd box = RFU signal strength

 

 

 

 

 

 

 

 

 

 

 

 

(peak height)
A7-PPT31-7 a Green PPT31-7
D881179 D2181] 018851 6000
amelogenin 4000
ll 2000
17
[103.75] M495]
6830 2576]
II!
[31952
162.67
101
A7-PPT31-7 a Yellow PPT31-7 .
D13S317 2000
D58818 ”3830 1500
1000

500

 

 

FIG. lZ—Sample TS1-7 (extracted with Chelex® and amplified with the Proﬁler PlusTM

amplification kit) exhibited a full single-source female proﬁle with additional activity.
Three allele calls, 13, I4, and 1 7, were observed at the D8S1] 79 locus. The I 7 allele fell

below the minimum threshold of150 RF Us for interpretation; therefore, this allele was
not considered. An additional allele (7) was present at the D5S818 locus. Since the base
pair size matches the I 7 allele at the D3Sl358 locus, this call may have resulted from

pull-up. It was not considered to be an allele or activity for this reason.

50

 

 

 

 

 

 

 

 

 

  

   

  
 
  

 

 

 

  

 

 

 

 

 

 

50 100 150 200 250 300 350 400
A6-PPTS16 7 Blue PPTS1-6
FGA 1000
D3Sl358 WA
500
-m-- 2-. JJi ..L. -2_. -2
[5E] E lst box = allele call
[131.72] [1 7535 224893 2nd box = base pair size
329 3rd box = RFU signal strength
191 , 23 (peak height)
267
A6-PPTSl-6 7 Green PPTSl-G
amelogenin D881 179 150°
1321511 D18851 1000
h 500
.. - -h 1 - 1 1
266
A6-PPTSl-6 7 Yellow PPTSl-6
D78820 800
D58818 33
200
9 ,
268.70
125
10
272.83
82

 

FIG. 13—Sample TS1-6 (extracted with Chelex® and amplified with the Proﬁler PlusTM
amplification kit) exhibited a single-source male proﬁle. With 3 uL enhancement, this
proﬁle was expected to be interpretable at all loci shown.

51

50 100 150 200 250 300 350 400
AS-PPT 81-5 6 Blue PPTS1-5

 

 

 

 

 

 

 

 

 

 

 

 

   

1000
D3Sl358
500
__.J.il- 4 - 1 A .
lst box = allele call
2nd box = base pair size
3rd box = RF U signal strength
(peak height)
A5-PPT81 -5 6 Green PPT81-5
amelogenin D8511” D2181] 018351 2000
1000
158.37
7
A5—PPTS1-5 6 Yellow PPTS15
0133317 073820 12:33
058818 1000
500

 

FIG. 14—Sample T S] -5 (extracted with Chelex® and amplified with the Proﬁler PlusTM
ampliﬁcation kit) exhibited a single-source male proﬁle with additional activity. Three
allele calls, 8, 10, and 13, were observed at the D1353] 7 locus. The 10 allele fell below
the minimum threshold of 150 RF Us for interpretation; therefore, it was classiﬁed as
activity and was not considered.

52

50 100 150 200 250 300 350 400
Bl1-PPT82-11 25 Blue PPT82-11

800
600
400
200

- ‘A A4 - A A J - A.“

lst box = allele call
2nd box = base pair size
3rd box = RFU signal strength

 

 

 

 

 

 

 

 

 

 

 

 

 

(peak height)
Bl1-PPT82-11 25 Green PPT82-11
. D881179 D18851 2000
amelogenin
1000
_ 4 l
[1 | [283.13]
410]
14
291.42
404
Bll-PPTSZ-H 25 Yellow PPT82-11
D1383]?
D58818 D78820 2000
1000

 

 

 

 

 

FIG. lS—Sample T 52-1 1 (extracted with Chelex® and amplified with the Proﬁler
PlusTM ampliﬁcation kit) exhibited a mixture of female DNA. The presence of three
alleles at two of the loci (D851] 79 and D1353] 7) and the imbalance of alleles at the
vWA, D2151], and D75820 loci indicate two donors with shared alleles consistent with

heredity by a full offspring. The ratio of contribution (female to female) is approximately
2:].

53

III|III‘IIIIIIIIIIIIIII'IIIIIII‘IIIIIII'III'IIIIIIIIIII|III|III.III'III'III‘III‘III

20 40 60 80 100 120 140 160 160 200 220 240 260 280 300 320 340 360 360 400

BB4¥W826 ZDBMB PPT826
vWA F G A from

D3Sl358 2000

1000

 

 

lst box = allele call
2nd box = base pair size
3rd box = RFU signal strength

 

 

 

 

 

     

(peak height)
eerp'rsz-e 20 Green PPTSZ-G
D2181] 913351 76000
Amelogenin D881 1 79 :40“
-2ooo
A b
Be-PPTSZ-G , 20 Yellow PP'rsz-e
Dl3S317 D7 '820 T°°°°
US$818 S 4000

 

12000

 

   

FIG. 16—Sample T 52-6 (extracted with Chelex® and amplified with the Proﬁler PlusTM
ampliﬁcation kit) exhibited a mixture of female and male DNA (D351 358, vWA, F GA,
amelogenin, D851] 79, D215] 1, and D1855] are depicted above). The presence of the 1’
allele at amelogenin, the imbalance of the X and Yallele at amelogenin, the presence of
three alleles at three of the loci (F GA, D851] 79, and D18551) and the imbalance of the
remaining alleles indicate two donors with shared alleles consistent with heredity by a
full offspring. The ratio of contribution (male to female) is approximately 2:].

54

While the isolation of embryonic/fetal DNA was indicated in results from both
extraction methods, the proﬁles generated from organically-extracted samples were
frequently deemed insufficient for comparison purposes or suitable only for limited
comparison. Signiﬁcantly less DNA was recovered from the organically-extracted
samples (see Statistical Estimates below) and there was a possible relationship between
unsuccessful organic results and the amount of DNA observed in the QuantiblotTM
procedure; partial proﬁle or no proﬁle results were more prevalent for samples that
contained less than the target 1.0 ng of DNA. With fewer interpretable alleles identiﬁed,
assuming many were attributable to the embryo/fetus; comparative power of the sample
was diminished.

Chelex®-Extracted and Proﬁler PlusTM-Ampliﬁed Samples

Data (electropherograms) from samples extracted with Chelex® and ampliﬁed
with Proﬁler PlusTM were examined for embryonic/fetal proﬁles (Tables 10—12). Two
samples, T81-6 (Fig. 13) and T81-9, generated single-source male proﬁles. One sample,
T81-5, produced a single-source male proﬁle with additional allelic activity detected that
fell below reporting threshold. Eight samples, T81-7 (Fig. 12), T82-10, T82-14, T83-A,
T83-B, T83-E, T83-J, and T83-V, generated single-source female proﬁles with
additional allelic activity that fell below reporting threshold. The following 25 samples
were mixtures of two females: T81-2, T81—3, T82-8, T82-9, TS2-11 (Fig. 15), T82-12,
T82-13, T82-15, T82-16, T82-17, T82-18, T82-19, T83-C, T83-D, T83-F, T83-I, TS3-
K, T83-M, T83-N, T83-O, T83-Q, T83-S, T83-T, T83-U, and T83-W. Nine samples

were mixtures of a male and female (Fig. 16), T81-4A, T81-4B, T82-1, T82-2, T82-3,

55

T82-4, T82-5, TS2-6 (Fig. 17), and TS2-20. Five samples, T81-l (Fig. 11), T81-8, T81-
10, T82-7, and T83-R, produced single source female proﬁles.

TABLE lO—Genetic proﬁle composition of T est Set 1 samples

 

 

2‘“ ‘1 I H " Chelex®
‘1 Sample Id# Proﬁler
, . PlusTM’l‘
T8] 1 SS—F
T512 . ' I . MX-F
TSl 3 MX-F
T81 4A ’ , .. MX-M ,
T81 4B MX-M
. .TSLS _, SST-MM),
T8] 6 SS-M
'. TS'I.7..’]".'. '33-F(A)
T81 8 SS-F SS-F (A) SS-F (A) SS-F
. (T8191, V. . .SSrM. (SS-1M, SS-F . SS-F”
T81 10 SS-F SS-F SS-F SS-F

*Chelex®-extracted and Proﬁler PlusTM-ampliﬁed samples

TChelex®-extracted and COﬁlerTM-ampliﬁed samples

IOrganically-extracted and Proﬁler PlusTM-ampliﬁed samples

§Organically-extracted and COﬁlerTM-ampliﬁed samples

88, single-source; MX, mixture; F, female; M, male; (A), allelic activity below reporting
threshold; empty ﬁeld, ampliﬁcation not conducted—no sample results

56

TABLE ll—Genetic proﬁle composition of Test Set 2 samples

 

Chelex® Chelex® Organic Organic
Sample Id# Proﬁler COﬁlerTM'l' Proﬁler PlusTM‘t COﬁlerTM§
PlusTM*
T82 1 MX-M MX-M SS-M
“ 1 -- "T52 '2‘ ' Mx-‘M ., . ~ WM - MX~M .
TS2 3 MX-M MX—M MX—M MX-M
TS2 5 MX-M MX—M MX-M
TS2 6 MX-M MX-M MX-M MX-M
T82 7 SS-F SS-F (A) SS—F (A)
T82 8 MX-F ‘ SS-F (A) MX—F
T82 9 MX-F MX-F
T82 10 , SS-F (A) SS—F MX-F
T82 11 MX-F SS-F (A) MX—F
T82 12 MX—F SS-F MX-F
T82 13 MX-F MX-F MX-F
T82 14 SS-F (A) SS-F (A)
T82 15 MX-F SS-F (A) MX—F
T82 16 MX-F SS-F (A) SS—F (A) SS-F
T32 17 MX-F 33—1: (A) SS-F (A)
T82 18 MX-F MX-F SS-F
T82 19 MX-F MX-F MX—F MX-F
.2 T82 20 i i , MX-M 7 MX—M

*ChelexG-extracted and Proﬁler PlusTM-ampliﬁed samples

IChelex®-extracted and COﬁlerTM-ampliﬁed samples

IOrganically-extracted and Proﬁler PlusTM-ampliﬁed samples

§Organically-extracted and COﬁlerTM-ampliﬁed samples

88, single-source; MX, mixture; F, female; M, male; (A), allelic activity below reporting
threshold; empty ﬁeld, ampliﬁcation not conducted—no sample results

57

TABLE 12—Genetic proﬁle composition of Test Set 3 samples

Chelex® Chelex® Organic + Organic
Sample ld# Proﬁler COﬁlerTM'l' Proﬁler PlusTM“ COfllerTm
Plusm“ .
T83 A SS-F (A) SS-F (A) SS-F
TS3'B SS-F (A) SS-F (A) SS-F
T83 C MX-F MX-F SS—F (A)
T33 D MX-F ' MX-F MX~F
T83 E SS-F (A) SS-F (A) MX-F
T83 F . MX—F MX-F MX-F
T83 l MX-F MX-F MX-F
T83 J SS-F (A) ' SS-F (A) SS-F (A) SS-F (A)
T83 K MX-F MX-F SS-F (A)
T83 M MX-F MX-F MX-F MX-F
T83 N MX—F MX-F MX—F MX-F
T83 O MX-F MX—F MX-F
T83 Q MX-F MX-F SS-F (A)
T83 R SS-F ‘ SS—F SS-F (A)
T83 S MX-F MX-F SS-F (A) SS-F
T83 T MX-F MX-F MX-F
T83 U MX-F MX-F MX-F MX-F
T83 V SS-F (A) SS-F (A) MX-F MX-F
T33 w MX-F MX-F SS-F (A) SS-F (A)

*Chelex®-extracted and Proﬁler PlusTM-ampliﬁed samples
lChelex®—extracted and COﬁlerTM-ampliﬁed samples
lOrganically-extracted and Proﬁler PlusTM-ampliﬁed samples
g‘Organically-extracted and COﬁlerTM-ampliﬁed samples
88, single-source; MX, mixture; F, female; M, male; (A), allelic activity below reporting
threshold; empty ﬁeld, ampliﬁcation not conducted—no sample results
Chelex®-Extracted and COﬁlerTM-Ampliﬁed Samples

Electropherograms from samples extracted with Chelcx® and ampliﬁed with
COﬁlerTM were examined for embryonic/fetal proﬁles (Tables 10—12). Three samples—

TSl-S, T81-6, and T81-9 are single-source male proﬁles. Results from 13 samples were

single-source female proﬁles with additional allelic activity that fell below reporting

58

threshold (T81-8, T82-7, T82-8, T82-11, T82-14, T82-15, T82-l6, T82-17, T83-A,
T83-B, T83-E, T83-J, and T83-V). Nineteen were mixtures of two females (T81-2, T8 1 -
3, T82-9, T82-13, T82-18, T82-19, T83-C, T83-D, T83-F, T83-I, T83-K, T83-M, TS3-
N, T83-O, T83-Q, T83-S, T83-T, T83-U, and T83-W). Nine were mixtures of a male
and a female (T81-4A, T81-43, T82-l, T82-2, T82-3, T82-4, T82-5, T82-6, and T82-
20). Six samples, T81-1, T81-7, T81-10, T82-10, T82-12, and T83-R, were single-
source female proﬁles.
Q_rg_anicallv-Extracted and Proﬁler PlusTM-Ampliﬁed Sarmales

Results for samples organically-extracted and ampliﬁed with Proﬁler PlusTM were
examined for embryonic/fetal proﬁles (Tables 10—12). Sample T82-l was a single-
source male proﬁle. Two, T81-4B and T81-5, were single-source male proﬁles with
additional activity detected which did not meet reporting threshold. Thirteen samples,
T81-2, T81-3, T81-8, T82-7, T82-16, T82-17, T83-C, T83-J, T83-K, T83-Q, T83-R,
T83-S, and T83-W, were single-source female proﬁles with additional activity detected
which fell below reporting threshold. Seventeen were mixtures of two females (T82-8,
T82-10, T82-11, T82-12, T82-13, T82-15, TS2-19, T83-D, T83-E, T83-F, T83-I, T83-
M, T83-N, T83-O, T83-T, T83-U, and T83-V). Seven were mixtures of a male and a
female (T81-4A, T81-7, T82-2, T82-3, T82-4, T82-5, and T82-6). Seven samples, TSl-
l, T81-6, T81-9, T81-10, T82-18, T83-A, and T83-B, were single-source female
proﬁles. No results were obtained for the following samples: T82-9, T82-14 and T82-

20.

59

Organically-Extracted and COﬁlerTM-Ampliﬁed Samples

Organically-extracted sample sets were ampliﬁed with COﬁlerTM and examined
for embryonic/fetal proﬁles (Tables 10—12). Two samples, T81-4B and T81-5, were
single-source male proﬁles with additional allelic activity which did not meet reporting
threshold. Samples T81-2, T81-3, T83-J, and T83-W were single-source female proﬁles
with additional activity detected which fell below reporting threshold. Five samples,
T82-19, T83-M, T83-N, T83-U and T83-V, were mixtures of two female proﬁles. Five
were mixtures of a male and a female proﬁle (T81-4A, T81-7, T82-3, T82-5, and T82-
6). Six samples were single-source female proﬁles (T81-1, T81-8, T81-9, T81-10, T82-
16, and T83-8). There were no results observed for the following samples: T81—6, T82-
], T82-2, T82-4, T82-7, T82-8, T82-9, T 82-10, T82-11, T82-12, TS2-13, T82-14, T82-
15, T82-17, T82-18, T82-20, T83-A, T83-B, T83-C, T83-D, T83-E, T83-F, T83-I, TS3-
K, T83-O, T83-Q, T83-R, and T3S-T.
Concordance of Proﬁles Between Ampliﬁcation Systems and Extraction Methods

Each Chelex®-extracted Proﬁler PlusTM-ampliﬁed sample was compared to the
same Chelex®-extracted COﬁlerTM-ampliﬁed sample at overlapping loci D3Sl358 and
D7 8820. Results were consistent for all samples that exhibited this information. The
same comparison was made between the organically-extracted Proﬁler PlusTM and
COﬁlerTM samples. This comparison was not conducted for the following samples based
on the lack of results for one or both ampliﬁcations: T81-6, T82-1, T82-3, T82-7, T82-8,
T82-9, T82-10, T82-11, T82-12, T82-13, T82-14, T82-15, T82-l7, T82-18, T82-20,
T83-A, T83-B, T83-C, T83-D, T83-E, T83-F, T83-I, T83-K, T83-O, T83-Q, T83-R,

and T83-T. There were no inconsistencies observed between the remaining samples that

60

exhibited results at the D3Sl358 and D78820 loci. All samples were examined for the
presence of additional alleles. None of the samples exhibited more than the expected
maximum of three alleles at any locus. Further comparisons of the collective proﬁle
information from the Chelex®-extracted Proﬁler PlusTM- and COﬁlerTM-ampliﬁed
samples and the organically-extracted samples were conducted. The alleles generated
from analysis of the Chelex®- and the organically-extracted samples were concordant,
however, not identical. The Chelex®- (Fig. 12) and organically— (Fig. 17) extracted
Proﬁler-PlusTM ampliﬁed T81-7 samples illustrate this point. The Chelex® T81-7 result is
a single-source female proﬁle with additional activity and the organic is a mixture of
female and male DNA. The Y allele at amelogenin, the 15 allele (activity) at D1885] ,

and the 16 allele at D381358 dropped out of the Chelex® sample.

61

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

50 100 150 200 250 300 350 400 450
AB-PPT81-7 9 Blue PPTS]-7
. FGA 3000
0331358 ‘WA 2000
1000
lst box = allele call
2nd box = base pair size
3rd box = RFU signal strength (peak height)
A8-PPTSl-7 9 Green PPT81-7
D1885] 6000
D8Sll79 D2181]
Amelogenin 4000
2000
103. 87
6273
A8-PPTS1-7 9 Yellow PPT81-7
D13S317 973320 1500
US$818 1000
500
11 1 1 11 1 l 1 1 1 1 1 1
272.80
295

   

FIG. l7—5ample T51-7 (extracted organically and amplified with the Proﬁler PlusTM
amplification kit) exhibited a mixture of female and male DNA (D351358, ame'IOgenin,
D851] 79). The presence of the Y allele at amelogenin, the imbalance of the X and Y

allele at amelogenin, the presence of three alleles at the D851] 79 locus and the
imbalance of the D351358 and D1855] alleles indicate two donors with shared alleles.

62

Identiﬁcation of Associated Samples

Based on the comparison of the sample proﬁle results (all loci—Proﬁler PlusTM
and COﬁlerTM) for both extraction methods, 16 different abortions were identiﬁed (Table
13). Some determinations were made utilizing results ﬁ'om only one extraction
procedure due to the lack of results for the other. Some were made based on results from
one ampliﬁcation system (Proﬁler PlusTM or COﬁlerTM) due to the lack of results from
the other. Figures 15 and 18 illustrate the association of two proﬁles ﬁ'om abortion

number 12.

TABLE 13—Associated samples from Test Sets 1, 2, and 3

 

 

3 T31-4A, T81-4B
5 T31-6
6 , j , _ T81-7
7 T81—8
.. 8. , . , ,. . _ . . T81-9 ,
9 T81-10
‘10 ' _ ,_ 7 ‘ ,,T32-'l,T32-2,‘T'32’-3,T324
11 T82-5, T82-6, T82-7
12 ' T32-8, T32-9, T82-10, T82—1 1, T32-12, T82-13, T32-l4
l3 T82-17, T82-18
14 ' ‘ ’ T82-19
15 TS2-20
. 16 i ' All samples ﬁom T83

63

50 100 150 200 250 300 350 400
BlGPPTSZ-lo 24 Blue PPT82-10

 

1500
0331358 100°
500
__...ue - - - 1 -

lst box = allele call
20d box = base pair size
3rd box = RF U signal strength

 

 

 

 

  

 

 

 

 

(peak height)

B10-PPT82-1O 24 Green PPT82-10
Dgsmg 021311 018351 :33:
amelogenin 2000
l 1000

A .

153 91

1889

Bio-PPTSZ-‘IO 24 Yellow PPT82-10
4000
D58818 D13S317 D78820 3000
2000
1000

 

 

 

 

 

 

FIG. 18—5ample T 52-10 (extracted with Chelex® and amplified with the Proﬁler
PlusTM amplification kit) exhibited a single-source female proﬁle with additional activity.
The presence of activity at one locus (D1353! 7) indicates two donors. The additional
activity allele at D351358 was due to pull-up and was not considered.

Comparison of Single-Source Female Proﬁles to Associated Samples

Results from four of the Chelex®-extracted Proﬁler PlusTM-ampliﬁed samples
were single-source female proﬁles (T81-l, T81-8, T81-10, and T83-R). T81-1
originated from the same abortion as T81-2. Results for sample T81-2, however,
revealed a mixture of females. Because of this, it was not possible to discern if T8 1 -1
could be attributed to the mother or the female fetus. T81-8 and T81-10 were the only
samples from two different abortions; therefore, no ﬁrrther comparison to aid in the
determination of the proﬁle source was possible. All of the samples ﬁ'om T83 were from
one abortion. Other samples from this abortion revealed mixtures of the same two female
proﬁles (T83-C, D, E, F, I, K, M, N, O, Q, S, T, U, V and W), therefore, no ﬁrrther
source determination was possible. Six of the Chelex®-extracted COﬁlerTM-ampliﬁed
samples, T81-l, T81-7, T81-10, T82-10, T82-12, and T83-R, were single-source female
proﬁles. The sources of T81-l and T83-R were not identiﬁable (see above). Samples
T81-7 and T81-10 were the sole samples from two separate abortions. No further
information regarding source was obtained. Single source female samples T82-10 and
T82-12 were from the same abortion; however, other samples indicated a mixture of
female DNA. The comparison did not further aid in source determination.

Results from six samples extracted with the organic method and ampliﬁed with
Proﬁler PlusTM were single-source female proﬁles. These included T81-1, T81-6, T81-9,
T81-10, T83-A, and T83-B. Comparisons of T81-1 , T83-A and T83-B to associated
samples were not helpful (previous paragraph). T81-6, T81-9, and T81-10 were the only

samples from three different abortions, so, there were no samples to compare.

65

Six of the organically-extracted COﬁlerTM-ampliﬁed samples were single-source
female proﬁles (T81-1, T81-8, T81-9, T81-10, T82-l6, and T83-S). Other samples from
the abortion which resulted in T82-16 were from a mixture of female proﬁles.

Peak Height Imbalance Between Loci

Samples that were extracted organically displayed more severe imbalances in
peak height between loci than those extracted with Chelex®. In the most extreme
example of this—organically-extracted Proﬁler PlusTM-ampliﬁed TSl-4B—the smaller-
sized loci D3Sl358, D881179, D58818, and amelogenin (base pair size ranges less than
171) had large peak heights near or exceeding the upper threshold for interpretation
(4500 RFUs), while the F GA, D1 8851, and D78820 loci (base pair size ranges greater
than 215) exhibited RFU values near or below the minimum interpretation guideline (150
RFUs). Comparison of Chelex®-extracted (Fig. 19) and the organically-extracted (Fig.
20) sample results for T81-4B illustrate imbalances of peak height between loci. The
highest peak height allele for the D58818 locus and the lowest peak height allele for the
D78820 locus were considered for both T81-4B samples. The lowest at D78820 for the
Chelex® extracted sample was 291 RFUs and the highest at D58818 was eight times
larger at 2477 RFUs. The lowest for the organically-extracted sample was 72 RFUs. The
highest was approximately 83 times larger at 5952 RF Us. The difference between the
allelic ratios of the two samples is approximately 10 fold. Further evidence for this
difference was observed upon comparison of single-source result Proﬁler PlusTM-
ampliﬁed samples where the alleles at the D58818 and D78820 loci were designated as
either heterozygous at each (peak height relationship must be at least 70%) or

homozygous at each. Chelex®-extracted samples ﬁt these criteria: T81-1, T81-4B, T8 1 -

66

6, T81-9, T81-10, T82-14, T83-A, T83-B, T83—E, T83-J, T83-R and T83-V. The
organically—extracted samples that ﬁt the criteria were as follows: T8 1 -l , T81-4B, TS3-
B, T83-C, T83-E, T83—F, T83-J, T83-K, T83-Q, T83-R, T83-S, and T83-U. Ratios were
calculated for the highest allele peak height observed at D58818 and the lowest at
D78820 for each. The average ratio of the Chelex®-extracted samples was 1 to 6 while

the average for the organically-extracted samples was 1 to 14.

67

III'III'IIIIIII'III'III'IIIIIIIIIII'III‘III|III|III‘III‘III‘III‘III‘III‘III'III‘III
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
E6-PPTS] ~48 59 Blue PPTS1-4B

vWA FGA 1 500

1000
500

D3Sl358 l

AAJJb11 111

 

 

 

 

lst box = allele call

2nd box = base pair size

3rd box = RFU signal strength
(peak height)

 

 

 

 

E6—PPTS] 4B 59 Green PPTSMB

-1 1 -11
13

109.35 149.52

2369

 

       

Amelogenin

E6-PF’T81-48 59 Yellow PPT81-4B

2000
1500
1000

D58818
1 500

A A- A - -1 1

 

 

FIG. l9—5ample T51-4B extracted with the Chelex® method and ampliﬁed with the
Proﬁler PlusTM amplification kit. The 1] allele at the D55818 locus had a peak height of
24 7 7 RF Us which was 8.5 times larger than the 13 allele at the D75820 locus which had
a peak height of 29] RF Us. All alleles from the major DNA contributor fell within
interpretational guidelines.

68

50 100 150 200 250 300 350 400 450
A5-PPTS1-4B 6 Elm PPTSl-4B

vWA FGA 3:83

2000
1000

   

D381358

 

lst box = allele call
2nd box = base pair size
3rd box = RFU signal strength (peak height)

   

 

A5-PP'TS1-48 6 Green PPTS1-4B

D881179 DZISll DISSSI 000

000
2000

        
 

Amelogenin

 

 

A5-PPTSl-48 6 Yellow PPTSl-48
000

000
2000

D58818

 

 

 

FIG. 20—Sample TS1-4B extracted with the organic method and amplified with the
Profiler PlusTM amplification kit. The 1 1 allele at the D55818 locus had a peak height of
5952 RF Us which was 83 times larger than the 13 allele at D75820 which had a peak
height of 72 RF Us. The 1] allele at D55818 exceeded the maximum threshold for
reporting (4500 RF Us). The 13 allele did not meet the minimum threshold for reporting
(150 RF Us). Enhancement of this sample to reduce the D55818 allele RF Us (1—4
seconds) and to increase the D75820 allele RF Us (3 uL preparation of amplified DNA)
was necessary but would require interpretation to be conducted over two
electropherograms.

69

Oﬁ‘ Ladder Alleles

The Genotyper® software assigned off-ladder allele designations (OL alleles) to
all of the peaks generated for organically-extracted, Proﬁler PlusTM-ampliﬁed samples
T82-1, T82-2, T82-3 (Fig. 21) and T82-4. No deﬁnitive conclusions could be made for
three of these, as TSZ-l, T82-2, and T82-4 were consumed for ampliﬁcation on the initial
attempt. The samples should have been re-injected immediately, or the ampliﬁed product
should have been re-prepared and injected. Neither option was available at the time of
analysis. Since sample T82-3 was not consumed, it was re-ampliﬁed with the Proﬁler
PlusTM kit. The peak pattern remained the same; however, the base pair sizes of the
alleles differed by approximately 0.5—1.5 base pairs between ampliﬁcations. Allele

designations were assigned for each of the peaks (Fig. 22).

7O

IIIIIIUIIll'lll'lll'lll'lll'lll'Ill'lll'lll'IIOIIII'III'II‘l'lll'IIQ'IIIIIII'IIIIIII'lll.ll
20 40 60 50 100 120 140 160 180 200 220 240 250 280 300 320 340 360 380 400 420 440
BZ-PPTSZG 16 BUB PPTSZ-3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

D381 358 2°°°
J 1000
4“- A A or :Hlele :2 _
lst box - allele call .
2nd box = base pair srze
3rd box = RFU signal strength
(peak height)
Bz-PPTsz-a 16 Green PPrsz-a
D1885] 3°°°
Amelogenin D881179 D213] 1 4000
2000
- [OT Ailelﬁ] [0L Allele-1’]
OL Allele ?
424
424
0L Allele '?
154 35
BZ-PPT82-3 16 Yellow PPTSZ-S D1353 l 7 D78820
r3000
D58818 3"”
71000
8 0L Allele ? or Angle . OL Allele 7
149 40 206.41‘
3268 l834
0L Allele 7 0L Allele .
153 91 218.64
1799 1330

 

FIG. 21—Sample T 52-3 (organically-extracted) with 0L allele designations. The
Genotjyper® software was not able to designate alleles for the D3Sl358, F GA, vWA,
amelogenin, D851 1 79, D21S11, D1855], D55818, D13S3I 7, and D7S820 loci due to the
.5—1.5 base pair shift of the alleles in comparison to the Profiler Plus TM ladder.

71

illllllllll'lll'lll'lilllllllIt'lll'lll'llI'lllllll'lll'llllIII.III|III'|II|IIIIll
20 40 30 30 100 120 I40 150 130 200 220 240 260 230 300 320 340 330 380 400
ALP-“32.3 23.18 P+T32-3

   

 

 

 

 

 

 

   

   

WA FGA i3°°°
V -2ooo
D381358 L1 000
lst box = allele call
2nd box = base pair size
3rd box = RFU signal strength
(peak height)
A1_P+T82-3 2 Green P-l-TSZ-a
D851179 D2181] . ooo
Amelogenin D1 8831 F000
A1_P+T32-3 2 Yelovr P+rsa-3 D138317
D75820
D58818 Fm”
1000

 

 

 

FIG. 22—Sample T S2-3 (organically-extracted) with allele designations. The sample
was re-amplified with the Profiler Plus TM kit. Upon comparison to the ladder, the

D3S1 358, F GA, vWA, amelogenin, D8S1] 79, D2151], D18S51, D55818, D1333] 7, and
D7S820 alleles were designated correctly.

72

Statistigil Estimates
A two-tailed t test using the separate variance estimate (Bachman and Paternoster,
1997) was used to determine whether a signiﬁcant difference in recovery of DNA existed

between extraction methods. The following were used to calculate the tom value and

degrees of freedom:
x. = 438.69 uL
xz = 70.85 uL
51 = 386.50
82 = 73.48

r11 = 50 samples extracted with Chelex®
r12 = 50 samples extracted organically

The average amount of DNA recovered from the Chelex®-extracted samples was 438.69
uL compared to 70.85 uL from organically-extracted. DNA recovery with Chelex® was
approximately six times higher than organic extraction. The calculated tom value was
6.54, with 55 degrees of freedom. The critical value of t with 55 degrees of ﬁ'eedom and
alpha value of .05 is 2.00. The calculated value of tom falls outside the critical region of
i200. Thus, the null hypothesis was rejected and a signiﬁcant difference in recovery of
DNA existed between extraction methods.

A 2 test (Bachman and Paternoster, 1997) for the difference between proportions
was applied-to the attempts made at extraction versus the actual recovery of a full genetic
proﬁle. The 2 test was used to determine whether a signiﬁcant difference existed

between extraction methods in obtaining full genetic proﬁles (Tables 7-9). The

following were used to calculate the Zobt and Gp1-p2 values:

131 = .96
p2 = .52
GP] p2 = .062

73

Ninety-six Chelex-, but only ﬁfty-two organically-extracted samples resulted in full
proﬁles. The associated sample proportions where .96 and .52, respectively. The pooled
standard error was calculated as .062. The calculated 20m value was 7.09. When using a
95% conﬁdence interval, the z scores of the critical region are :1: 1.96. The calculated
value of zobt falls outside the critical region of i1 .96. Therefore, the Chelex® and organic

extraction methods differ signiﬁcantly in recovery of full genetic proﬁles.

74

DISCUSSION

Historical Issues and Procedures Developed

Sampling from early abortion materials collected within two to twelve weeks of
conception for criminal DNA paternity testing poses multiple problems. The ability to
visually discern embryonic or fetal tissue from maternal tissue is generally not possible
before eight weeks. The Michigan State Police Lansing Laboratory frequently utilized
random tissue sampling techniques during these time periods, which oﬁen failed to detect
embryonic/fetal proﬁles due to the presence of overwhelming amounts of uterine tissue.
Karger et a1. (2001) developed a procedure to identify chorionic villi among recovered
tissues for DNA testing in an effort to ensure embryonic/fetal proﬁle results while saving
time and money. The tissue was formalin-ﬁxed and suspended in parafﬁn blocks for
microsectioning and microscopic identiﬁcation of the chorionic villi, followed by DNA
testing. Six samples from one abortion—one with identiﬁed chorionic villi—were
ampliﬁed targeting ten STR loci, nine of which were CODIS core loci. Proﬁles were
obtained from the six samples—one was a mixture and ﬁve were single source. Four of
the samples only produced partial proﬁles (4 loci) and were consistent with the mother.
One sample was a mixture of alleles consistent with the mother and an offspring.
Analysis of the sample in which chorionic villi were identiﬁed resulted in a full proﬁle
consistent with an offspring of the mother and putative father. Karger et a1. (2001)
referenced extraction methods from two papers (Iwasa et al. (1997) and Klintschar et a1.
(1999)) each of which utilized a different extraction method—Chelex® and modiﬁed

alkaline lysis respectively, although it is not clear which of these Karger et al. (2001)

75

used. Likewise, their sampling source—tissue from prepared slides or parafﬁn blocks—
was not outlined. There was no indication of quantity and quality of recovered DNA.
Finally, since few samples were tested, it was not clear if the level of success could be
reproduced.
DNA Recovery and Isolation of Embryonic/Fetal DNA

The goals of the research presented here were to test the microscopic
identiﬁcation of chorionic villi across a broad scope of samples, to compare the Chelex®
and organic extraction methods in their ability to generate the highest quantity of DNA
from the embedded tissue, and to attempt ampliﬁcation of the recovered DNA with
Proﬁler PlusTM and COﬁlerTM kits in an effort to generate full CODIS proﬁles
representative of the embryo/fetus. The method which resulted in the most complete
embryonic/ fetal DNA would provide greater discriminatory power upon comparison with
suspect samples. Upon comparison of the quantiﬁcation data from samples comprised of
formalin-ﬁxed embryonic/fetal tissue and maternal decidua it was found that signiﬁcantly
greater quantities of DNA were recovered using the Chelex® method (Tables 1—3) than
the organic method (Tables 4—6). Further comparison of proﬁle data following
electrophoresis revealed signiﬁcantly more full proﬁles were produced with Chelex®
extraction than with the organic extraction method (Tables 7—9). A relationship appeared
to exist between the quantity of DNA recovered and the generation of a full proﬁle.
Many of the organically-extracted samples did not yield enough DNA for ampliﬁcation
with both kits; therefore, the maximum number of loci was nine instead of thirteen.

Data were compared from each extraction method. The organically-extracted

samples exhibited more severe imbalances of peak height between loci. The most

76

illustrative example of this was sample TS 1-4B, in which the ratio of the lowest peak
height from the D78820 locus to the highest peak height from the D58818 locus was 1:83
(Figure 20). The same ratio for the Chelex-extracted sample T81-4B was 1:8 (Figure

19). The average ratio of the lowest peak height at D78820 to the highest peak height at
D58818 was 1:6 for the Chelex®-extracted samples and 1:14 for the organically-extracted
samples. Reasons for imbalance of peak height between loci include DNA degradation
and/or the presence of a PCR inhibitor (see Explanation of Chelex® Success below). The
difference in average peak height ratio obtained for the Chelex®-extracted samples was
less drastic than that of the organically-extracted samples, indicating DNA of higher
quality.

The electropherograms from Chelex®- and organically-extracted samples were
examined for the presence of embryonic/fetal proﬁle information. Six classiﬁcations of
proﬁle results were observed—single-source female, single-source female with additional
activity, single-source male, single-source male with additional activity, mixture of
females, and mixture of a male and a female. All classiﬁcations conﬁrmed the presence
of embryonic/fetal alleles except single-source female proﬁles, in which case the alleles
may be attributed to the embryo/fetus or to the mother. It was impossible to determine
the donor (embryo/fetus or mother) of the single-source female proﬁles, unless other
associated abortion samples (Table 13) contained male DNA. In those instances it was
clear that the male DNA was attributed to the embryo/fetus and the female DNA to the
mother. All of the single-source female proﬁles were compared to associated abortion
samples if they existed (Table 13), but these yielded no further information for

discernment.

77

The presence of embryonic/fetal proﬁle information was indicated in the majority
of samples; however, the usefulness of the proﬁle information could not be determined in
many circumstances. Results classiﬁed as single-source female proﬁles with activity
could contain interpretable proﬁle information from either the embryo/fetus or the
mother. If the activity portion of the proﬁle was attributed to the embryo/fetus, the
information would be useless for associative purposes in a criminal proceeding.
Incomplete mixtures of two females could contain the embryonic/fetal proﬁle as the
major donor (complete) and the maternal proﬁle as the minor component (incomplete) or
the reverse. Associations would be less discriminating if the minor contributor was the
embryo/fetus.

Single-source female proﬁles may contain exclusively embryonic/fetal DNA or
maternal DNA; no conﬁrmations were possible. If these proﬁles were conﬁrmed as
embryonic/fetal, ay be very useful for association if was not determined. The proﬁles
may be attributed to either the embryo/fetus or the mother. the proﬁle would be useful
only if it was attributed to the embryo/ fetus.

Concordance of Samples Between Ampliﬁcation Systems and Exugction Methods

Allelic information obtained from Chelex®-extracted samples was concordant
with that of the organically-extracted samples in all cases. However, in many instances
the allele calls at each locus were not identical, but were consistent with heredity
relationships between an individual and its full offspring in varying proportions. This
result is likely due to the tissue sampling technique. Tissue sections were taken from
adjacent areas of the same parafﬁn block in these experiments, and obtaining an identical

ratio of embryonic/fetal tissue to maternal decidua was improbable due to the variation

78

present in each block. Allelic information at shared loci (D3Sl358, D7S820, and
amelogenin) between Proﬁler PlusTM and COﬁlerTM ampliﬁed samples was also
concordant. Less variation was observed with DNAs between ampliﬁcation sets than
between extraction sets, as these were ampliﬁed from the same extracted sample.
Identiﬁcation of Associated Samples

All electropherograms were compared and proﬁle relationships discerned in order
to identify related samples among the 50 cassettes analyzed. It was determined that the
three test sets of formalin-ﬁxed, parafﬁn-embedded embryonic/fetal tissue and maternal
decidua were comprised of materials from sixteen abortions (Table 13). Since this
discernment was possible, it is further evidence of the utility of the research methods in
obtaining useful allelic information for interpretation. The combination of sample data
from each abortion most often generated more proﬁle information—increasing the
comparative power of the samples for paternity determination. Electropherograrns from
TS2-11 (Fig. 15) and TS2-10 (Fig. 18), which are associated abortion samples, illustrate
this point.
Explanation of Superior Chelex® Results

Explanations for the greater success observed with Chelex® extraction compared
to organic extractions include more effective removal of inhibitors such as iron in heme
molecules present in blood, decrease in nuclease activity catalyzed by magnesium, and
reduction in formalin-protein and formalin-DNA interaction. According to Wilson
(1997), inhibitors interfere with PCR ampliﬁcation (inactivation of the DNA
polymerase), degrade or capture nucleic acids, and interfere with cell lysis in extraction.

It is possible that the Chelex® process is more successful in removing common PCR

79

inhibitors such as porphyrin compounds in blood (Walsh et al., 1997) than is the organic
preparation. Hemoglobin, a protein containing heme, is found in blood and is released by
proteinase K (used in both extraction procedures of this experiment) during the extraction
process. Chelex® beads can bind iron (carried by heme) separating it ﬁom the DNA
(Walsh et al., 1997), whereas the organic procedure may not effectively reduce or remove
enough iron to facilitate complete PCR ampliﬁcation.

Romero et al. (1997) stated that historically, DNA from formalin-ﬁxed tissues
does not amplify well due to degradation. During the ﬁxation process, DNA degrades
rapidly, the effects of which can cause poor ampliﬁcation of larger sized loci. Chung et
al. (2004) observed poor ampliﬁcation for loci within the 300 to 500 base pair (bp) range
in samples exposed to formalin. Shibata et al. (1994) found that PCR ampliﬁcation of
formalin-ﬁxed tissue was optimal between target lengths of 80 and 170 bp. In the
research presented here, nine of the Proﬁler PlusTM and COﬁlerTM amplicons exceed 170
bp in length; however, the effects of degradation were exhibited in loci greater than 200
bp (considered larger sized loci). It is possible that the use of mini-STR sets, with
smaller amplicon sizes of approximately 100 bp (Butler et al., 2003), could be utilized to
reduce or eliminate the effects of degradation (W iegand and Kleiber, 2001). The use of
multiplex systems such as Proﬁler PlusTM and COﬁlerTM (used in this research) require
fewer ampliﬁcations of the DNA sample and are therefore less labor intensive than mini-
STR sets. However, the loss of data for larger loci at the minimum threshold (150 RFUs)
and smaller loci at the maximum threshold (4500 RFUs) is a risk when using the
multiplex systems. In this research, the differences in peak height of alleles at small

versus large loci of the Chelex®-extracted samples were less pronounced than in samples

80

extracted organically. Samples extracted with Chelex® contained more interpretable
allelic information at more loci than did samples extracted organically. Both extraction
processes include digestion with tissue lysis buffer that contains EDTA, which binds
magnesium, blocking it from activating nucleases that destroy DNA. The iminodiacete
ions, attached to plastic Chelex® beads, chelate (bind) more of the remaining divalent
metal ions. The beads are collected at the bottom of the extraction tube by centrifugation,
separating them from DNA in the solution, further reducing degradation in these samples.
During the ﬁxation process, exposure of tissues to formalin results in the
formation of methyl bridges (cross-links) between the amino groups of purine and
pyrimidine bases (DNA) and proteins, and also between proteins (Brutlag et al., 1969;
Feldman, 1973; Kieman 2000; Moerkerk et al., 1990; Romero et al., 1997). Cross-
linking may occur in many different conﬁgurations, comprised of primarily linked
proteins or of a combination of DNA and proteins, and both may reduce input DNA for
PCR. This reduction may be purely mechanical, chemical, or a combination of both.
The organic extraction method relies on phase separation of organic solvents and aqueous
components and is effective due to the hydrophobic affinity of proteins and hydrophilic
afﬁnity of DNA. The formation of long cross-linked chains of proteins could cause
mechanical interference during phase separation, trapping DNA molecules in the organic
and interface layers. Likewise, cross-linked molecules containing DNA and proteins may
have a hydrophobic afﬁnity, chemically interfering with phase separation and carrying
DNA molecules into the organic and interface layers. In both circumstances, the upper
aqueous layer would be collected, and the interface and organic solvent layers, containing

some DNA, discarded. In this research, samples extracted organically recovered

81

signiﬁcantly less DNA than those extracted with Chelex®. The Chelex® extraction
method does not require separation; therefore more DNA may remain in the extraction
solution.

The digestion of tissue prior to either extraction method was accomplished with
tissue lysis buffer containing Tween 20 (detergent) and proteinase K to break open cells
and denature proteins. Cross-linked proteins caused by formalin exposure may not be as
easily denatured with proteinase K, possibly reducing the quantity of available DNA for
ampliﬁcation. The Chelex® extraction method utilizes a boiling step (100°C) to denature
proteins and break open cells. This additional denaturing of proteins and subsequent
release of DNA from cells could have increased DNA recovery. According to Overton
and McCoy (1996), temperatures of 75°C are known to disrupt cross-links between
formalin and DNA, which may also have increased input DNA for PCR.

Data Anomaly Observed

In this study, four samples—TS2-1, T82-2, T82-3 and TSZ-4——extracted
organically and ampliﬁed with Proﬁler PlusTM exhibited off-ladder allele (OL allele)
designations for many or most of the alleles (Fig. 21 ). According to Applied Biosystems
(1997), variation commonly occurs between samples injected with the same capillary.
However, variation causing measurement errors greater than i .5 base pairs results in the
designation of OL allele assignments. Six sample injections of the Proﬁler PlusTM ladder
were attempted. The ﬁrst two (PPLADDER and PPLADDER-2) failed. These were
injected nearest samples T82—1 , T S2-2, T82-3 and T82-4. Allele peak heights for both
injections fell below the minimum threshold (150 RFU) for interpretation, therefore,

could not be used. PPLADDER also exhibited 0L allele designations for many of the

82

alleles due to failure of the ROX internal size standard. The ROX peaks came off at
higher scan numbers consistently and the resolution of the peaks became progressively
broader throughout the electrophoresis. According to Applied Biosystems (1997) this
problem was most likely due to syringe malfunction—polymer did not ﬁll the capillary
before injection (Applied Biosystems, 2006). This could occur if air bubbles were
present in the syringe, if the pump arm was not aligned correctly, or if the capillary ﬁtting
was malfunctioning (Applied Biosystems, 2006).

Samples TS2-1, T82-2, T82-3, and T82-4 exhibited ROX peaks which came off
at lower scan numbers consistently throughout the electrophoresis. According to Applied
Biosystems (1997) this problem occurs when water is present in the syringe. This can be
avoided if the syringe is primed correctly with polymer to help remove water before the
ﬁnal polymer ﬁlling. The aforementioned problems worked themselves out by the sixth
injection. Under normal circumstances those six samples would have been re-injected
immediately; however, the 310 instrument was not available. Alleles present in samples
T82-1, T82-2, TS2-3, and T82—4 were not designated upon comparison to PPLADDER-
3, PPLADDER-4, PPLADDER-5 or PPLADDER-6 due to inaccurate sizing of the ROX
peaks. The samples all exhibited allele peak shifts of +0.5—1 .5 base pairs. Applied
Biosystems (1997), recommends re-injection of samples that contain 0L alleles above
minimum reporting threshold to verify reproducibility. If the OL alleles are reproduced
after re-injection, re-ampliﬁcation is recommended and reproducibility would conﬁrm the
presence of a true 0L allele. As was mentioned previously, re-injection of the
aforementioned samples was not possible due to equipment constraints. At a later date,

sample T82-3 (the only sample with the appropriate quantity of DNA remaining for

83

ampliﬁcation) was re-ampliﬁed and analyzed. Results revealed acceptable internal size
standard peaks and allele call designations (Fig. 22), none were reproduced as OL alleles.
Implementation of Chelex® Extraction Method for Embryonic/Fetal Tissue in the
Forensic Laboratog

The research presented utilized the following combination of methods: formalin-
ﬁxation, parafﬁn-embedding, microscopic identiﬁcation of chorionic villi, sampling of
the chorionic villi, xylene deparafﬁnisation, digestion (tissue), Chelex® or organic
extraction, quantiﬁcation, ampliﬁcation with Proﬁler PlusTM and COﬁlerTM ampliﬁcation
kits, electrophoresis, and analysis of the data. Since Chelex® extraction proved superior to
organic extraction, it in combination with the aforementioned methods was adopted as
standard operating procedure (SOP) and implemented at the Michigan State Police
Lansing Laboratory. Validation was completed in February 2004 and technical review
accepted it in May of 2004. This was a simple process as it involved only the comparison
of extraction methods (all other procedures were previously validated) and minimal
expense for reagents. Since May of 2004, numerous cases have been successfully
adjudicated in Michigan Circuit Courts.

Six of these cases were analyzed from the two year period since implementation.
Of the six, ﬁve had associated known samples ﬁ'om the mother and putative father.
Associations to the putative father were made on all of these. One of the cases was
analyzed in J une of 2006 and known samples have not been received by the submission
date of this research. However, the case had deﬁned results which indicated the maternal
proﬁle and the embryo/fetal proﬁle. Data from these cases were included to illustrate the

types of proﬁles that were generated—all identifying information has been removed.

84

Case 1

Sample 1 (cassette containing formalin-ﬁxed, parafﬁn-embedded embryonic/fetal

tissue and maternal decidua from an abortion) generated a mixture proﬁle with the most

embryonic/fetal alleles (Table 14A). Sample 3 was a single-source female proﬁle

consistent with the mother. Samples 2 and 4 produced mixture proﬁles which exhibited

some embryonic/fetal alleles. Upon comparison with the mother and putative father,

sample 1 was consistent with a full offspring of the mother and putative father (Table

14B). The allelic mismatch at D18S818 was most likely due to a mutation (either

paternal or maternal). Both parents have a 17 allele as does the offspring and the

offspring’s 15 allele is one repeat greater than the additional paternal allele and one

repeat less than the additional maternal allele.

TABLE l4A—Genetic data at 13 CODIS core loci for Case 1

Loci
D3 S l 3 58
vWA . ,
FGA
Amelogenin ,

D8Sll79 V

1321311
1318351
D53818 ,
D13S317
' 1373820
r1101
TPOX
CSFIPO
13163539

Sample 1

16,17
14,17,(19)
(19),20,28
,. X ,
(101,13
27,30.2,(312)
15,17
12‘
8,11
8,a A
7,(9),9.3
8,10,a
11
9,12

Sample 2

16,07)
17,19
19,20

X
10,13

302,312

16,17
12
11
8

9.9.3

8,11
A

9,12

Sample 3

16
17,19
19,20

. X
10,13
302,312

A

12

11

8
9,9.3
8,11

A

9,12

Sample 4

16,(17)
(14),17,19‘
19,20
f -X .
10,13
81,302,312
16,17
.. 12 _
11,a

8,a "
9,9.3,a
8,ll,a

11

9,12

( ), allelic activity of a minor contributor; a, additional activity detected that failed to
meet reporting standards; comma, separates multiple alleles present; period, indicates an
allele with deletion of one or more bases

85

TABLE l4B—Genetic data at 13 C ODIS core loci for Case 1

Mother Putative Discemed
Loci Father Offspring Proﬁle
D3Sl358 16 15,17 m,l7
vWA 17,19 14,15 14g
FGA 19,20 20,28 E28
amelogenin I X X,Y X
D8Sll79 10,13 12,13 13
D2181] 30.2,31.2 27,28 27,3_0;
D18851 16,17 14,17 15*,17
D58818 12 8,12 12
D13S3l7 11 8,13 8,ﬂ
D78820 8 8,10 8
THOl 9,93 7,8 1%
TPOX 8,1 1 8,10 8,10
CSFl PO 1 l 7,1 l l l
7 D168539 9,12 9,12 9,12

underlined, allele contributed by mother, bold, allele contributed by father; *, mutational
event suspected

Case 2

Sample 1 generated a mixture proﬁle of female DNA with the most
embryonic/fetal alleles (Table 15A). Sample 3 was a single-source female proﬁle
consistent with the mother. Sample 2 did not generate adequate allelic information for a
useful comparison. Sample 3 produced a single-source female proﬁle. Upon comparison
with the mother and putative father, samples 1 and 3 were consistent with a full offspring

of the mother and putative father (Table 15B).

Loci

D3Sl358

vWA
FGA

Amelogenin
D881179

,[D2131 1'
D1885]
D58818 ,
D13S317

"D7S820

THOl

', TPOX . .
c31=11>o
,- ‘D163539 _

i ' Sample 1"

16,18
14,06)
20,21
X
13,15
27,29,(31)
12,14,a
‘1 1,0 2)

(8),11,13

8,11;a_.
6,(8),9

. 3,11 _ .
10,11

' Sample 2 I

18
A
A
X
13

A
ND

ND

ND

_ ND .

ND

TABLE ISA—Genetic data at 13 CODIS core loci for Case 2

“Sample 3‘

18
14,16
20,21

X

13
29,31

A
11,12

8,a

A

8,9

A

ND

11

Sample 4

16,18
14
20,21
X
13,15
27,29
3
11
a

a _

l 6,9

8,11
ND

,_ w_11

( ), allelic activity of a minor contributor; a, additional activity detected that failed to
meet reporting standards; comma, separates multiple alleles present; period, indicates an
allele with deletion of one or more bases; ND, no alleles detected for this locus

87

TABLE 15B—Genetic data at 13 CODIS core loci for Case 2

Mother Putative

   
   

Discemed
Loci , Father Offspring Proﬁle
D3Sl358 18 14,16 16$
vWA _ _ 14,16 14,18 14
FGA 20,21 20,21 20,21
melogemn , X ' X,Y
D881 179 13
D18851
D13S317
D7S820
THOl
TPOX
CSFIPO
D168539 11 11,12 11

underlined, allele contributed by mother; bold, allele contributed by father
Case 3
Samples 1, 2, and 3 generated mixture results of female and male DNA (Table

16A). Sample 4 produced a single-source male proﬁle. Upon comparison with the
mother and putative father, sample 1 generated the most complete embryonic/fetal proﬁle
and was consistent with a full offspring of the mother and putative father (Table 16B).
The allelic mismatch at CSFl P0 was most likely due to a mutation (maternal or
paternal). Both parents and the offspring have a 13 allele, and the offspring’s 11 allele is

one repeat greater than the additional maternal allele and one repeat less than the

additional paternal allele.

Loci

D3Sl358
vWA

FGA

Amelogenin
D881179
‘D21811
D1885]
D58818
D138317
' ,D7S820 _

TH01

(TPOX ,
csrrpo
" . D168539) ..

3" Sample 1

16,18
16,17
20,23,a
X,Y
10,(13),16
29,31.2,a
12,15
10,12,(l3)
9,13,a
10,1 1‘
6,7
9,11
13
. .1 1,1,3

Sample 2

16417)]8
16,17,a
20,23
X,Y
10,(13),16
29,31.2,a
12,15
10,12,(1 3)
9,13,a
10,a
6,7
9,1 1
13

,. 11,13,

TABLE l6A—Genetic data at 13 CODIS core loci for Case 3

Sample 3

16,18
16,17
ND
X,Y
10,(13),16
A
ND
10,12
ND
ND
A
ND-
ND

..ND, _.,

Sample 4

16,18
16,17
20,23
X,Y
10,16
29,312
12,15
10,12,a
9,13
11,a
6,7
9,1]
A
(11,a

( ), allelic activity of a minor contributor; a, additional activity detected that failed to
meet reporting standards; comma, separates multiple alleles present; period, indicates an
allele with deletion of one or more bases; ND, no alleles detected for this locus

89

TABLE l6B—Genetic data at 13 CODIS core loci for Case 3

Mother Putative Discerned
Loci Father Offspring Proﬁle
13331358 16 16,18 1_6,18
' vWA 17,19 16,19 16,11
FGA 20,23 22,23 20,23
amelogenin ’ x X,Y X,Y
D8Sll79 10,13 12,16 19,16
D21311 ‘ 29,322 30,312 _2_9_,31.2
1318351 15,19 12,20 12,15
1353818 12,13 10,13 fro,_1_2_
D133317 9,12 12,13 2,13
1373820 11 10 10,11
TH01 6,7 6,7 6,1
TPOX 11 9,11 V9,_1__1_
CSFlPO 10,13 12,13 11*,13
D168539 , ,11 ‘ 11,12 _ 11

underlined, allele contributed by mother; bold, allele contributed by father; *, mutational
event suspected

Case 4

Sample 1 generated a partial single-source female proﬁle (COﬁlerTM only) (Table
17A). Sample 2 generated a mixture of male and female DNA (Proﬁler PlusTM only).
Sample 3 produced a mixture of female DNA (full proﬁle Proﬁler PlusTM and COﬁlerTM).
Sample 2 contained the most complete embryonic/fetal allelic information even though it
was a partial proﬁle including only Proﬁler PlusTM data. Upon comparison to the mother
and putative father samples, sample 2 was consistent with at least one ﬁrll offspring of the
mother and putative father (Table 17B). The presence of the Y allele and additional
allelic presence and balance in sample 2 indicates that this may have been a multiple
pregnancy. Sample 1 was consistent with a full offspring of the mother and putative

father. Since the overlapping D3Sl358 allele matches the Discerned Offspring Proﬁle 1,

90

it most likely reﬂects the same source. Sample 3 was consistent with the mother and a
full offspring of the mother and putative father.

TABLE l7A—Genetic data at 13 CODIS core loci for Case 4

Sample 1 Sample 2 Sample 3
Loci
D3Sl358 l7 17 17
vWA ND 15,16 14,15,16
FGA ND 22.2,a 19,222,232
amelogenin X X,(Y) X
D881179 ND 8,12,16 8,12,16
‘ D2181] ND 29,30 29,30
D1885] ND ND l3,15,a
, D58818 , ND 11,13 11,12,13
D138317 ND 9,13 9,13
1373820 9 ’ ND ‘ 9,10
TH01 5,6 ND 5,6,9.3
TPOX 8,12 ND 8,12
CSFIPO 12 ND 12,14
D168539 11,12 ND ,, 11,12

( ), allelic activity of a minor contributor; a, additional activity detected that failed to
meet reporting standards; comma, separates multiple alleles present; period, indicates an
allele with deletion of one or more bases; ND, no alleles detected for this locus; alleles
detected in Sample 1 are from the COﬁlerTM ampliﬁcation only; alleles detected in
Sample 2 are from the Proﬁler PlusTM ampliﬁcation only

91

TABLE 17B—Genetic data at 13 C ODIS core loci for Case 4

Mother Pirtative Discerned Discerned

Loci Father Offspring Offspring

Proﬁle 1 Proﬁle 2

D3Sl358 17 15,17 17 mg
vWA 14,15 14,16 15,16 15
FGA 19,232 2122.2 222m UK
Amelogenin x X,Y x x
D8Sll79 12,16 8,15 8,3 8,]_2__
f 1321311 30 29,31 29,19 29,10
D18S51 13,15 12,20 UK UK
' "053818 12,13 11,13 rr,_1_:_l'_ UK
D133317 9,13 11,13 2,13 UK

' D7S820 9,10 9,10 9 UK .
TH01 6,9.3 5,9.3 5,6 UK
TPOX _, 8 8,12 8,12 UK
CSFIPO 12,14 11,12 12 UK
__.D1.68539 _ .. .1132... .5 11 .¥ ., 11.1.2. _ . -UK

underlined, allele contributed by mother; bold, allele contributed by father

Case 5

Sample 1 was a single-source female proﬁle and was consistent with a full

offspring of the mother and putative father (Table 18).

92

TABLE 18—Genetic data at 13 CODIS core loci for Case 5

Sample 1' " ' ‘ Mother Putative ' ' DiScerned
Loci Father Offspring
Proﬁle
D3Sl358 15,16 15,16 16 1_5_,16
vWA 14,17 17,18 14,17 14,11
FGA 23 19,23 20,23 23
Amelogenin X X X,Y X
D831179 13 13 13,14 13
1321311 29,30 29,30] _ 28,29 29,39
D18351 14 14,15 14,15 14
f D53818 11,12 11,12 ‘ 7,11, 11,12 ‘
D13S317 12,13 13 11,12 12,13
" D7S820 10,11 10 8,11. _1_Q,11
TH01 9,9.3 6,9 9.3 9,93
(TPOX [8,11 8 " 8,11 , 8.11.,_
CSFIPO 10 10 10,12 10
’ .D168__5_39 ‘_1.1,12.,___ . V, 1112 e __ 9,12 .6 , 11.12

( ), allelic activity of a minor contributor; a, additional activity detected, but it failed to
meet reporting standards; ND, no alleles detected at this locus; underlined, allele
contributed by mother; bold, allele contributed by father
Case 6

Sample 1 generated a single-source male proﬁle with additional activity (Table
19). Samples 2 and 3 generated mixtures of female and male DNA and were comprised
of higher ratios of maternal to embryo/ fetal DNA. Sample 3 was predominantly maternal
and was used to compare to sample 1. Proﬁles of the mother and offspring were

discemable. Known samples would of course be necessary for conﬁrmation. Alleles

contributed by the father were able to be determined also.

93

TABLE l9—Genetic data at 13 CODIS core loci for Case 6

Sarnplel Sarnple'2 Sample 3' . Discerned

Loci Offspring
Proﬁle

D331358 16,17,a 17 (16),17 16,11
vWA 14,15 14,18,a l4,(15),18 14,15
FGA 20,23 " 20,22,a 20,22,(23) 20,23
Amelogenin X,Y X,Y X,Y X,Y
D8Sll79 12,13,a 12,(13) 12,(13) £13
. D21311 30,312 30,(31.2) 30,(31.2) 39,312
D18351 13,132,a 12,13,a 12,13,(13.2) 13,132
1353818 ' 11 ‘ 11 ’ 11 ’ 11
D133317 8,12 (8),11,12 (8),11,12 8,12
1373820 10,1 1* 10,a, 10,(_1 1) " ,_1_l_3_,11
TH01 6,7 6,(7),9.3 6,(7),9.3 _6_,7
TPOX ‘ 8 . 8 _ 8' ' ‘ 8
CSFIPO 12 11,12 11,12 12
D168539" _ ., 12. _,12,13.. __.12,13. .12

( ), allelic activity of a minor contributor; a, additional activity detected that failed to
meet reporting standards; comma, separates multiple alleles present; period, indicates an
allele with deletion of one or more bases; underlined, allele contributed by mother; bold,
allele contributed by father
Recommendations for Handling Aborted Materials in Criminal Cases

A detailed protocol for handling rape complaints resulting in pregnancy is
necessary. The law enforcement ofﬁcer, doctor performing the abortion, forensic
pathologist, DNA testing laboratory scientist, and prosecutor have crucial roles in
evidence collection, preservation, and viability. Establishment of a clear method—
agreed upon by all potential parties involved—would logically aid in proper handling of
rape resulting in pregnancy cases. It would seem that the education of law enforcement
personnel would be of the utmost importance as these individuals are usually responsible

for evidence transfer facilitation. A list of recommendations for law enforcement

personnel has been developed (Fig. 23).

94

 

 

10.

. Obtain written consent or a search warrant to allow conﬁscation of the

aborted embryonic or fetal materials.
Contact the medical facility performing the abortion to request the
following:

A. Written results of the estimated term of pregnancy from an

ultrasound procedure

B. The abortion procedure used

C. That the tissue be placed into a clean, sterile container and secured

with evidence tape, and initialed and dated by the collector

D. That the tissue be frozen fresh and not placed into ﬁxative.

E. Obtain written chain of custody information at the time of receipt
Transport the materials frozen or maintain at the coldest temperature
possible—use a cooler with ICC if the facility does not provide something
comparable.

If the term of pregnancy is 12 weeks or less or unknown, contact the
facility that performs autopsies for your agency and request an examination
of the materials.

If the term of pregnancy is greater than 12 weeks take the materials
directly to the forensic laboratory that services your jurisdiction (skip #7).
Arrange to transport the processed fetal/embryonic tissue ﬁom the
pathologist to the forensic laboratory.

Notify the forensic laboratory of the contents so that they are stored
appropﬁately(ﬁozen) prior to and following analysis.

Obtain victim known buccal swabs or known whole blood in an EDTA
(purple-topped) blood collection tube and transport it to the laboratory with
a request for comparison to the aborted embryonic or fetal materials.
Obtain a warrant or written consent for the suspect’s known buccal

swabs.

Collect buccal swabs from the putative father and transport them to the
laboratory for analysis with a request for comparison to the aborted
embryonic or fetal materials.

 

 

FIG. 23—List of recommendations for law enforcement personnel. If followed, these
ensure proper handling, documentation, and analysis of the embryonic/fetal tissues.

Recommendation 1 ensures that the embryonic/fetal materials have been legally

seized and will be admissible in court proceedings. The medical facility will demand this
for release of the materials as well. Recommendation 2A is necessary to determine
which facility will receive the materials for ﬁrrther processing (see 5 and 6). 2B provides

information as to the possible state of the materials after the abortion procedure, which

95

may assist the pathologist in screening. 2C will aid in tracking of the materials for court
admissibility purposes. 2D will reduce the risk of additional DNA degradation (Butler,
2005). Recommendation 3 is necessary because this may not be recorded at the medical
facility, and the ofﬁcer may be creating the sole record of the transfer (necessary for
admissibility in court). The fourth recommendation for frozen transport will reduce the
risk of additional DNA degradation (Butler, 2005). Recommendations 5, 6, and 7 ensure
that the appropriate professional is receiving the materials for preparation for DNA
analysis. Recommendation 8, if followed by the receiving agency, ensures that the
materials are identiﬁed and stored correctly to avoid additional DNA degradation or
inhibition. Recommendations 9 and 11 provide known samples ﬁom the mother and
putative father for DNA analysis and comparison to the embryonic/fetal tissues. The
known samples are necessary for determination of paternity and calculation of the
supporting likelihood ratio. Recommendation 10 ensures court admissibility of the
suspect’s DNA proﬁle.
Conclusions

The samples tested contained varying proportions of forrnalin-exposed,
microscopically identiﬁed chorionic villi and maternal tissues ﬁom abortion procedures.
All samples were subjected to xylene deparafﬁnisation, extracted using either the
Chelex® method or the organic method, ampliﬁed with Proﬁler PlusTM/COﬁlerTM kits and
analyzed using capillary electrophoresis. Samples extracted with the Chelex® method
resulted in signiﬁcantly higher quantities of DNA than samples extracted organically.
Likewise, the Chelex®-extracted samples exhibited signiﬁcantly higher quality DNA with

less severe peak height imbalances between loci (presumably caused by degradation or

96

inhibition) and more complete proﬁle information. The source (embryo/fetus or mother)
of the proﬁle was not discernible in some circumstances (i.e. single-source female
proﬁles and single-source female proﬁles with additional activity). Mixture results
exhibited embryonic/fetal proﬁle information, however, the usefulness of the information
is dependant on the ratio of the allelic contribution from the donors determined by the
presence and balance of alleles. Comparisons were conducted on all test set sample data
and resulted in the identiﬁcation of sixteen different abortions. This further illustrates the .
utility of the microscopic examination and identiﬁcation of chorionic villi. Experiments
including known maternal and paternal samples are necessary for identiﬁcation of the
contributors to each sample and, in turn, the likelihood of obtaining full embryonic/fetal
genetic information from analysis. Results of data to date ﬁ'om implementation of the
procedures with Chelex® extraction have been favorable. In ﬁve of six cases, association
of embryonic/fetal results with putative father samples was possible. Based on the results
of this research, both extraction methods isolated STR DNA proﬁle information from
abortion materials; however, the Chelex® extraction method was superior in DNA

quantity and quality recovered for criminal paternity comparison.

97

APPENDICES

98

APPENDIX A

STATUTORY CIRCUMSTANCES FOR CSC I and CSC II

99

Victim under 13 years of age.
Victim at least 13 but less than 16 years of age, AND any of the following:

> Perpetrator is a member of the same household as victim;

> Perpetrator is related to victim by blood or afﬁnity to the fourth degree; OR,

> Perpetrator is in a position of authority over the victim, AND used this
authority to coerce the victim to submit.

Sexual act involves the commission of any other felony.

Perpetrator aided or abetted by one or more other persons, AND either of the
following:

> Perpetrator knows or has reason to know that the victim is mentally
incapable, mentally incapacitated, or physically helpless, OR
> Perpetrator uses force OR coercion.

Perpetrator armed with a weapon OR an article fashioned so as to lead a person to
reasonably believe it is a weapon.

Perpetrator uses force or coercion AND causes personal injury.

Perpetrator causes personal injury and knows or has reason to know the victim is
mentally incapable, mentally incapacitated or physically helpless.

Victim is mentally incapable, mentally disabled, mentally incapacitated or physically
helpless and one of the following:

> Perpetrator is related to the victim by blood or afﬁnity to the fourth degree;
OR,

)> Perpetrator is in a position of authority over the victim and used this authority
to coerce the victim to submit.

Perpetrator is an employee, contractual employee, OR volunteer with the Department
of Corrections AND knows that the victim is under its jurisdiction (CSC II only).

Perpetrator is an employee, contractual employee, OR volunteer with a private vendor
that operates a youth correctional facility AND knows that the victim is under the
jurisdiction of the Department of Corrections (CSC 11 only).

Perpetrator is an employee, contractual employee, OR volunteer with a county or the
Department of Corrections AND knows that the victim is prisoner or probationer
under the jurisdiction of the county (CSC II only).

100

Perpetrator is an employee, contractual employee, OR volunteer with the facility in
which the victim is detained awaiting trial OR hearing OR in which the victim is
committed as a result of having been found responsible for committing an act that
would be a crime if committed by an adult (CSC 11 only).

101

APPENDIX B

STATUTORY CIRCUMSTANCES FOR CSC III and CSC IV

102

Victim is at least 13 but less than 16 years of age (CSC 111 only).

Victim is at least 13 but less than 16 years of age AND the perpetrator is ﬁve OR
more years older than the victim (CSC IV only).

Perpetrator uses force OR coercion.

Perpetrator knows OR has reason to know the victim is mentally incapable, mentally
incapacitated, OR physically helpless.

Perpetrator is related to the victim by blood OR afﬁnity to the third degree AND
sexual penetration OR contact occurs under circumstances not otherwise prohibited
by the CSC Act.

Perpetrator is a mental health professional AND sexual contact occurs during OR
within two hears after victim was patient OR client of perpetrator AND victim was
not the perpetrator’s spouse (CSC IV only).

103

APPENDIX C

PROTOCOL FOR THE PREPARATION OF FORMALIN-FIXED PARAFFIN-
EMBEDDED EMBRYONIC/FETAL TISSUE AND MATERNAL DECIDUA FOR
DNA EXTRACTION

104

Sample Preparation

1. Observe microscope slides which correspond to parafﬁn blocks identiﬁed as
containing chorionic villi and locate the areas with villi for sampling.

2. Cut away the outer layer of the parafﬁn block with a sterile blade and obtain a small
segment of tissue (approximately 2—4 mm3) from the block and place in a labeled
microcentrifuge tube.

Deparaffinisation

1. Add 1.0 mL of xylene to each tube (perform in chemical hood) to remove parafﬁn
wax.

2. Incubate for 30 minutes at room temperature and centrifuge for 2—5 minutes at
15,300 RCF. Remove and discard the supernatant into the appropriate waste
container.

U)

Repeat steps 1 and 2 for a total of 2 xylene washes.
4. Add 1.0 mL of ethanol to each tube to remove xylene.

5. Centrifuge for 2-5 minutes at 15,300 RCF. Remove and discard the ethanol into the
appropriate waste container.

6. Repeat steps 4 and 5 for a total of 2 ethanol washes.
7. Dry tissue for 10—20 minutes at 15—20 in. Hg in a Hetovac vacuum apparatus.
Digestion

1. Prepare Tissue Lysis Buffer (fresh daily). Add 200 nL of Tissue Lysis Buffer to
each tube.

Tissue Lysis Buffer

1.89 mL TE“ Buffer (10 mM, pH 7.5)
10 11L Tween 20 (0.5%)

m 11L Protdpwe K (20 ng/mL)
2.0 mL Total

2. Incubate at 37°C overnight. Centrifuge for 5 minutes at 15,300 RCF.

105

1.

Collection and Purification

Remove the solution (sample) from the digestion tube and transfer to a Centricon-
100TM concentrator assembled according to the manufacturer’s instructions (sample
reservoir with attached retentate vial was ﬁtted to a ﬁltrate vial). Discard any
remaining tissue.

Centrifuge for 30—60 minutes or longer if necessary (until most of the liquid passes
through ﬁlter into the ﬁltrate vial leaving the ﬁlter membrane moist) at 2000 RCF.
Discard ﬁltrate.

Add 2.0 mL of TE buffer to the sample reservoir. Centrifuge 30—60 minutes or
longer if necessary at 2000 RCF. Discard ﬁltrate. Repeat once.

Remove and discard the ﬁltrate vial, invert the sample reservoir with retentate vial
attached and centrifuge for 3 minutes at 1000 RCF.

Transfer the retentate (concentrated sample) directly from the Centricon-IOOTM
concentrator to a labeled microcentrifuge tube. The samples are ready for extraction.

106

 

APPENDIX D

PROTOCOL FOR CHELEX EXTRACTION OF DNA

107

 

Chelex® Extraction Protocol

1.

2.

Add 20 uL of 5% Chelex® solution to the sample tube and vortex brieﬂy.
Incubate at 56° C for 30 minutes then vortex at high speed for 5—1 0 seconds.

Incubate in a boiling water bath for 8 minutes then vortex at high speed for 5—1 0
seconds.

Centrifuge for 3 minutes at 15,300 RCF. Samples are ready for quantiﬁcation.
For short-term storage place at 2—8°C on the Chelex® beads. Repeat steps 2, 3 and 4

before use. For long term storage, transfer the supernatant ﬁ'om the Chelex® beads
to a new tube and freeze at -20°C.

108

APPENDIX E

PROTOCOL FOR PHENOL/CHLOROFORM/ISOAMY L ALCOHOL (ORGANIC)
EXTRACTION OF DNA

109

 

Organic Extraction Protocol

1.

2.

Add 200 11L of phenol/chloroform/isoamyl alcohol to the sample tube.
Vortex until a milky emulsion is produced.
Centrifuge in microcentrifuge for 5 minutes at 15,300 RCF.

Assemble a Centricon—lOOTM concentrator according to manufacturer’s guidelines
(sample reservoir with attached retentate vial must be ﬁtted to a ﬁltrate vial).

Draw off the top aqueous layer of solution and transfer to the concentrator.
Centrifuge for 30—60 minutes or longer if necessary (until most of the liquid passes
through ﬁlter into the ﬁltrate vial leaving the ﬁlter membrane moist) at 2000 RCF.
Discard ﬁltrate.

Add 2.0 mL of TE buffer to the sample reservoir. Centrifuge 30—60 minutes or
longer if necessary at 2000 RCF. Discard ﬁltrate. Repeat once.

Remove and discard the ﬁltrate vial, invert the sample reservoir with retentate vial
attached and centrifuge for 3 minutes at 1000 RCF.

Transfer the retentate directly from the Centricon-l 00TM concentrator to a labeled
microcentriﬁrge tube. The samples are ready for quantiﬁcation.

llO

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111

 

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