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THESIS

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ICHIGM TATE WEBSITY LIBRARIES

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This is to certify that the

thesis entitled

IDENTIFICATION OF HUMAN DNA FROM BED SHEET
LINENS USING THE POLYMERASE CHAIN REACTION
(PCR) FOR DQAl AND POLYMARKER LOCI

presented by

Marilyn Joyce Galvan

has been accepted towards fulfillment
of the requirements for

M.S. degree in Criminal Justice

\

 

Major p fessor

Date JJIJ/il/Cl7

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

LIBRARY
Michigan State
University

 

 

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PLACE IN RETURN BOX
to remove this checkout from your record.
TO AVOID FINES return on or before date due.

 

DATE DUE

MTE DUE

DATE DUE

 

”£2239“

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1/98 chlHC/DfloDmpGS—p.“

IDENTIFICATION OF HUMAN DNA FROM BED SHEET LINENS
USING THE POLYMERASE CHAIN REACTION (PCR) FOR
DQAl AND POLYMARKER LOCI
By

Marilyn Joyce Galvan

A THESIS
Submitted to
Michigan State University
in partial fiilfillment Of the requirements
for the degree of

MASTER OF SCIENCE

School of Criminal Justice

1998

ABSTRACT
IDENTIFICATION OF HUMAN DNA FROM BED SHEET LINENS
USING THE POLYMERASE CHAIN REACTION (PCR) FOR
DQAl AND POLYMARKER LOCI
By

Marilyn Joyce Galvan

The identification of missing persons in the absence of fingerprints, dental X-rays
or direct identification may be necessary for criminal or civil litigation. Retrieval of
shed skin cells from bed sheets may serve as a source of DNA for comparison
purposes to aid in the identification of adults and children. The purpose of this study
was to evaluate the feasibility of recovering DNA fiom Skin cells shed on bed sheets
as a source for genetic information in missing person cases.

Shed skin cells fi'om nineteen human subject’s bed sheets were collected using an
adhesive roller and the DNA extracted by phenol/chloroform with ethanol
precipitation. Ten of these subjects were re-evaluated using vacuum collection and a
second adhesive retrieval. DNA from these subjects was captured using a Centricon®
100 microconcentrator in place of ethanol precipitation. The two extraction methods
were compared with respect to: 1) Efficiency of recovery 2) Contamination between
each subject retrieved 3) Amplification and genetic typing. Subject DNA from cheek
cells were used as a standard for comparison.

Results of this study demonstrated that cells collected from bed Sheets offer a rich
source of DNA for genetic testing and higher yields of DNA were Obtained with less

contamination using adhesive as a collection substrate.

Copyright by
Marilyn Joyce Galvan
1998

All things are possible to him who believes.

Mark 9:23

iv

ACKNOWLEDGMENTS

I would like to thank the Michigan State Police DNA Laboratory for their time and
assistance throughout this project. I am particularly grateful to Charles Bama for the
use of the State Police facility, his input and patience throughout the entire course of
this study. I am also deeply grateful to Dr. Mohammad A. Tahir who gave his time
and shared his expertise to teach me valuable techniques I will use now and in the
fiiture.

Throughout the course of my graduate studies, Dr. Jay A. Siege] has been a source
of guidance academically for myself and continues to work tirelessly to expose his
students to as many of the forensic disciplines as possible.

My research would be incomplete if it were not for the sincere, conscientious
cooperation of all my human subjects who chose to participate in this study. Due to
their cooperation and patience, data was produced which may be of benefit for years
to come.

Finally, my words cannot describe the constant support of Bill and Tim, my two

special guys, who adjusted their lives for me, while Mom went back to school.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................... vii

LIST OF FIGURES .......................................................................... viii

INTRODUCTION .............................................................................. 1

CHAPTER 1

History of DNA Variable Regions and Literature Review ................................. 4

CHAPTER 2

Factors Afi‘ecting DNA Amplification ......................................................... 9

CHAPTER 3

Materials and Methods ......................................................................... 12

CHAPTER 4

Results ........................................................................................... 19

CHAPTER 5

Conclusions and Recommendations .......................................................... 42

APPENDICES
Appendix A - Adhesive Roller Method ............................................... 47
Appendix B - Vacuum Retrieval Procedure ........................................... 49
Appendix C - Ethanol Precipitation Extraction Method ............................ 51
Appendix D - Centricon® Extraction Technique ................................... 53
Appendix E - Slot Blot Procedure for Skin Cell Quantitation .................... 55
Appendix F - Yield Gel for Buccal Cell Quantitative Analysis .................... 58
Appendix G - Reverse Dot Blot Method for Genetic Typing ..................... 60
Appendix H - Reagents and Supplies ................................................. 64
Appendix I - Glossary ................................................................. 70

LIST OF REFERENCES .................................................................... 72

vi

LIST OF TABLES

Table 4 - 1 First Adhesive Retrieval DNA Quantities .................................. 26
Table 4 - 2 DNA Quantities From Vacuum Retrieval Procedure ..................... 31
Table 4 - 3 DNA Quantities From Second Adhesive Retrieval Method ............ 32
Table 4 - 4 Compared DNA Quantities -

Vacuum Retrieval to Adhesive Retrieval .................................. 33
Table 4 - 5 Subjects Mouth DNA Quantities ............................................ 41
Table 5 - 1 Percent of Subjects Yielding Cells and DNA .............................. 43

vii

LIST OF FIGURES

Figure 4 - 1 Subject Data From First Adhesive Retrieval ............................... 20
Figure 4 - 2 Subject Data From Questionnaire ........................................... 21
Figure 4 - 3 Subjects Additional Data ..................................................... 22
Figure 4 - 4 First Group Bed Sheet DNA by Slot Blot ................................ 23
Figure 4 - 5 Second Group Bed Sheet DNA by Slot Blot ............................. 24
Figure 4 - 6 Third Group Bed Sheet DNA by Slot Blot ............................... 24

Figure 4 - 7 Subjects 1,2,4 and 5 Mouth and Bed Sheet DNA Amplifications ...... 27
Figure 4 - 8 Subjects 3,6 and 7 Mouth and Bed Sheet DNA Amplifications ........ 28
Figure 4 - 9 Subject 4 Re-Amplified DNA from Adhesive Retrieval ............... 29
Figure 4 - 10 Subjects 1,12,13,16 Re-Amplified DNA from Adhesive Retrieval. . .29
Figure 4 -11 Slot Blot of Vacuum Retrieval and Second Adhesive Extraction. ..30
Figure 4 -12 Mouth and Vacuum Amplification of Subjects 1-5 ...................... 33
Figure 4 -13 Mouth and Vacuum Amplification of Subjects 6,7,12,13 and 16 . ....34
Figure 4 -14 Second Adhesive Amplification of Subjects 12 and 16 .................. 34

Figure 4 -15 Subjects 1-7 and 12, Mouth and Vacuum Retrieval
DQAl Results .................................................................. 35

Figure 4 -16 Subjects 12,13 and 16 Mouth, Vacuum and Adhesive
DQAI Results ................................................................. 36

viii

LIST OF FIGURES (cont’d)

Figure 4 -17 Interpretation of DQAl Results Vacuum and Adhesive ................. 37
Figure 4 -18 Subjects 1-6 Mouth and Vacuum Polymarker Results ................... 38
Figure 4 -19 Subjects 7,12,13 and 16 Polymarker Results .............................. 38
Figure 4 -20 Interpretation of Polymarker Results Vacuum and Adhesive ........... 39
Figure 4 - 21 Subjects 1 through 6 Mouth Swab DNA .................................. 40
Figure 4 - 22 Subjects 7 through 19 Mouth Swab DNA ................................ 41
Figure 5 - 1 Order of Vacuum Specimens Showing Secondary Alleles .............. 44

Figure 5 - 2 Order Of Second Adhesive Specimens With Secondary Alleles ........ 45

ix

INTRODUCTION

A variety of methods have been relied on in the identification of missing persons.
Visual confirmation, dental records, forensic anthropology and fingerprints are used
to associate missing persons to unidentified bodies. Recent advances in DNA
technology have provided a new tool for human identification. Alec J. Jefli'eys first
applied DNA based typing techniques for human identification in the forensic setting
in 1985. Taking advantage of unique sequences referred to as variable number
tandem repeats (VNTR), Jeffiies successfirlly identified the donor of a semen
sample in a brutal rape homicide. This simple application signaled an explosion in the
application of DNA typing techniques for human identification.

Forensic laboratories are constantly challenged to develop information data from
biological samples. Minute quantities and sample degradation due to specimen age
and environmental insult are not unusual. The invention of a method to replicate
DNA sequences in large quantities by KB. Mullis in the 1985 revolutionized
molecular biology techniques and is commonly referred to as the Polymerase Chain
reaction (PCR).There are three advantages in applying PCR to forensic applications:
1) High molecular weight DNA is not a specimen requirement 2) Quantity Of input
DNA is only one to five nanograms and 3) Multiple genetic loci can be co-amplified

, simultaneously. The Polymerase Chain Reaction gained rapid acceptance in

developing methods for human identification as many of the most difficult obstacles
experienced in forensic biology were overcome.

The Polymerase Chain Reaction is a simple method which replicates short DNA
sequences of interest in vitro . The sample (target DNA), acts as a template for
replication of the specific DNA sequences. The PCR process occurs involves three
basic steps: 1) Separation of the DNA double strands into single strands by high
temperature (94°C) incubation 2) Lowering Of the temperature to allow binding of
complementary DNA sequences (primers) to flank the DNA area of interest and
3) Extension of the primers with the addition of nucleotides using the target
sequence as a template. This process is repeated for 32 cycles in standard forensic
procedures and yields millions of copies of the target area Of interest fiom originally
minute quantities Of DNA.

Since DNA technology has been accepted within the forensic community and the
criminal justice system, assessment of PCR inhibition, DNA stability and recovery of
various sources of DNA have been ongoing. From initial testing of nucleated blood
cells and semen, it was found that all human nucleated body cells Of sufficient quantity
and quality would give consistent DNA genetic types regardless Of specimen age.
Nasal secretions (Tahir, et al., 1995), single hair roots (Hochmeister, et al.,l995),
bone fragments (Lee, et al., 1991, Blake, et al., 1992), saliva (Presley, et a1. 1993),
finger secretions from fingerprints, internal body organs and external human skin
(Chen, et al., 1996), all have been found to give DNA types which correlate to
reference samples from the same individual such as white blood cells or buccal cells

(Thomson, et al., 1992) which line the inner cheek.

Cells from the outer layer of the human epidermis have been typed from adhesive
used for collecting gunshot residue by Torre and Gena in 1996. Adhesive has also
been used as a support medium impregnated with blood for high molecular weight
DNA retrieval (Stein, et al., 1996). There is no documented study to date which has
set out to investigate the feasibility of DNA retrieval fi'om shed human skin cells using
different support media and an optimum method of extracting the cells for PCR
amplification.

This study compares two procedures in which shed Skin cells fiom human subjects
were collected from bed sheet linens and extracted by two different methods. The
first method utilized an adhesive roller as a collection device and phenol/chloroform
with ethanol precipitation for the first extraction method. The second method used a
hand vacuum for collection onto moistened filter paper and a second adhesive
retrieval substituting Centricon® 100 micro concentration to capture the extracted
DNA.

Subjects were given no additional instructions regarding sleeping habits. Only
duration of exposure and the method of cell collection was intentionally varied. The
subjects Shed skin cells were then compared to buccal cheek cells as a reference for
genetic typing of six polymorphic DNA regions using the Polymerase Chain
Reaction. The goals of this study were to determine the feasibility of using shed skin
cells as a source of DNA for human identification and to evaluate the collection

systems and DNA extraction protocols described in this study.

Chapter 1

History Of DNA Variable Regions And Their Use In Forensic Science

The presence of blood group substances which caused immune reactions between
one individual and another have been Observed since the 19th century. In 1900,
Karl Landsteiner discovered that individuals of blood type A have antibodies which
precipitate the red blood cells of blood group B. In later experiments, it was shown by
Morgan and Watson that the difl‘erence between the group A and group B substances
found on the red cells of these individuals was due only to the replacement of an
OH-group in type B individuals or by a CHsCO-NH-group in the type A group. By
the mid-1940’s, Karl Landsteiner and AS. Wiener had found that 85% of the white
population possess another red cell antigen termed Rho, after the Rhesus monkey it
was discovered in. This discovery led to a hypothesis proposed by RA. Fisher and
RR. Race in which three separate genes were responsible for the red cell antigens
found on the surface of human red cells known as the Rh system.

In 1952, Dausset Observed adverse reactions between patients who had received
multiple blood transfirsions when blood components were being infused. In 1964,
Terasaki and McClelland introduced testing in which identification of a variety of
specific genetic differences between individuals could be Observed. By 1967, it was

shown that these differences in compatibility between cellular tissues of one individual

compared to another belonged to the same genetic system. This system was termed
Human Leukocyte Antigen or HLA to denote it’s genetic expression on the human
white blood cell (Miller, et al., 1981). Rejection of transplanted organs and tissues
and destruction of certain blood components is a result of HLA incompatibility due to
the extreme variability from one individual to another (Perkins, HA, 1997).

A concise review of DNA and genome variation for the forensic biologist was
presented by Fowler, et al. in which the human genome and it’s polymorphic nature
was described. The DNA double helix molecule consists of approximately 6 billion
nucleotides found within the human genome. These units are distributed within each
DNA double helix. Each pair of DNA chains are bound by hydrogen bonds. A pair of
chromosomes consists of two DNA molecules in which proteins (histones) are bound
to maintain the DNA structure. There are 46 chromosomes, or 23 pairs in each human
nucleated cell. The DNA chains are paired and coiled to form a double helix structure.

Certain areas found within the human chromosomes are known to code or dictate
the function of specific proteins used in physiological firnctions. These regions of the
human genome are called genes which consist of nucleotide bases in particular
sequences The individual variations within the gene are responsible for difl‘erences
physically as well as chemically throughout the human body and are called alleles.
Thus, variation due to nucleotide bases in certain locations within the human DNA
helix are responsible for traits such as hair color.

Approximately 20 to 30% of all human DNA is highly repetitive or reproduced in
multiple copies. The exact functions of these repetitive sequences is not always

known and appear to be distributed more frequently, seemingly at will, within the

individual’s genetic constitution. With the development of molecular biology
techniques, forensic scientists have been able to exploit the variability of the human
genome and develop techniques for human identification. The most discriminating
DNA test continues to be RFLP, the method proposed by Jefii'eys et al. In this
method, variation of DNA lengths due to repetitive nucleotide sequences are used for
inclusion or exclusion of the victim or suspect.

Vlfrth knowledge of the HLA system and the advent of PCR in the forensic
laboratory, a rapid PCR method was developed to use the variability of this system
along with five other locations known for their ability to discern individuals. Previous
investigation of sequences of nucleotides, consisting of a phosphate, a sugar molecule
and a base (adenine, thymine, guanine or cytosine) within the human DNA molecule
were reported by Slightorn, Blechl and Smithies in 1980. In their report, the complete
nucleotide sequence of two human genes were reported fi'om a Single individual. This
area known as hemoglobin G gammaglobin (HBGG), suggested an exchange can
occur between the two DNA chains at this genetic site on the chromosome designated
as number 11. Their findings also strongly suggested that DNA polymorphisms due to
addition, deletion or base molecule substitutions are quite common in the human
population.

In 1984, Yamamoto et. al, were able to reproduce the human LDL receptor.
Known for it’s influence in low density lipoprotein disease or familial high cholesterol,
they were able to identify the nucleotide sequence by reproducing the specific area in
question on chromosome 19. A year later, group-specific component (Gc) genetic

sequence was investigated due to it’s Vitamin D-binding capabilities. By combining

human liver DNA with mouse cells, the entire coding sequence of the human GC gene
was obtained (Yang, et al., 1985).

In 1989, a test method, reverse dot blot, described by Saiki, Walsh, Levenson, and
Erlich was reported for sequence-specific segments of DNA which were bound to a
nylon membrane. Using UV light, thymine bases were bound within the DNA chains
to the nylon. By tagging the primers used in the PCR process, visualization with a
streptavidin-horseradish peroxidase conjugate was possible to produce a colorimetric
reaction which was simple in laboratory practice and convenient compared to
dimculties with the use of radioactive visualization. This technique was applied to the
HLA-DQAI location of chromosome 6, which was of interest in forensic analysis
due to the extreme variability previously observed and became the first test utilizing
the polymerase chain reaction for forensic use made commercially available by the
Cetus Corporation in 1990.

The same year, Helmuth et al. reported fi'equencies of the I-ILA-DQAI location
of chromosome 6 in the US. Caucasian, U.S. Black, Southeast Asian, Japanese and
various Hispanic populations along with isolated Indonesian, New Guinea, Australian,
Bedouin and Nigerian populations. In their study, it was found that the discriminating
potential of the HLA-DQAI site was well-suited for use in forensic and paternity
determinations. A year later, Walsh et al. published the results of five forensic
laboratories which assessed the ability of the reverse dot blot for DQAl from blind
trials using typical forensic specimens such as blood, hair, semen, buccal swabs and

postcoital samples. The test laboratories had little to no PCR experience or exposure

to the reverse dot blot method. Of 180 samples studied, 178 samples were reported
and all were correctly typed.

By 1992, firrther validation studies had been performed on over 250 cases that had
been tested. The PCR method had the clear advantage of utilizing small or degraded
samples. Bloodstains on cloth, vinyl, leaves and paper, exposed to a variety of
environmental insults such as microorganisms, sunlight and chemicals such as bleach
and gasoline produced successfirl genetic typing without false negative or positive
reactions (Corney, et al., 1991).

To date, the reverse dot blot method continues to be utilized in laboratories as an
easy, rapid test for genetic typing in forensic and paternity determinations. Six genetic
markers (HLA-DQAI, LDLR,GYPA, HBGG, D788 and GC) are able to be
co-amplified simultaneously by the polymerase chain reaction (Budowle, et al., 1995).
These variable regions are then able to be typed by binding amplified target DNA
segments to complementary probes on membrane strips. With the addition of color

conjugate a visible blue color is produced (AmpliType® Userguide, 1995).

 

Chapter 2

Factors Affecting DNA Amplification

The polymerase chain reaction is a powerful tool in the forensic laboratory,
particularly with degraded specimens or minute quantities. However, it has not been
without problems. Damage to the DNA chains at the positions where primers anneal
will have an adverse effect on the PCR (Akane, et al., 1993). Humid conditions
enhance the growth of bacteria which produce nucleases that damage the DNA
molecule. Found in bacteria, plants and animals, nucleases are used by bacteria as a
means of self-repair and protection from genetic alteration. This enzymatic activity
profoundly effects the integrity of samples which have started to decompose by
destroying the target DNA sequence.

The type of body tissue used for DNA determination can be an indication of
suitable DNA recovery. Skin, blood and bone marrow are known as rich sources of
relatively unadulterated DNA and some tissues such as brain cortex and lymph nodes
are more usefiIl than liver, spleen and cardiac muscle. The degradation of DNA within
these tissues is dependent upon post-mortem factors such as elevated temperatures at
the death scene or disease of the deceased (Kobilinsky, L., 1992).

Chemicals have also been found which, when extracted along with nuclear DNA,

may inhibit the PCR. The exact mode of inhibition has not been fully investigated, yet

10

some chemical contaminants are known for their inhibitory tendencies when
amplifying the DNA molecule. The dye indigo, found in blue denim can be inhibitory
when DNA is extracted directly from dark blue denim, whereas lighter blue denim has
been found to be less inhibitory (Del Rio, et al., 1996). An extraction method called
Chelex® has produced successful amplification results for light and dark blue denim
fabric. This procedure involves the use of Chelex® reagent at a pH >_lO which
denatures the DNA double helix molecular structure. Because of the alkaline pH, the
DNA will remain denatured, thus limiting the sample to PCR based methods.

A variety of extraction protocols have been investigated and documented to
provide the forensic analyst with a successful protocol when working with particular
types of specimens. One classical extraction protocol utilizes Sodium Dodecyl Sulfate
(SDS), and Proteinase K to digest the body fluid. Phenchhloroform/rsoamyl is then
used to purify the DNA and precipitation is accomplished by the addition of ethanol.
This method has been found to be successful for amplification of blood impregnated
on a sweatshirt, wool sweater, floor linoleum and carpet (Corney, et al., 1991).

A successful and rapid method is commonly used for both RFLP and PCR
specimens that achieves concentration of the DNA and purifies it from potential or
known contaminants. By the use of centrifugal force and a series of washes using
Tris-EDTA Bufi’er, the contaminants are filtered through pores designed to retain the
DNA molecules while smaller particles are collected below in a retention device
(Centricon® Userguide, 1995).

Many forensic extracts exposed to feces, soil or water contain phenol groups

which bind proteins by forming hydrogen bonds. Bovine serum albumin (BSA), 3

11

protein itself, is known to act as a scavenger of such phenol-containing compounds.
The BSA binds to the phenol portion of the contaminant to inhibit inactivation of
proteins necessary for the PCR. Biological fats (lipids) can also bind to bovine serum
album which can compete with the enzyme Taq DNA polymerase.

BSA is also known to bind excess Proteinase K in vitro. Proteinase K is added to
break the protein bonds of the biological sample. Though added in measured
amounts, an excess can result due to endogenous and added protease. The enzyme is
then available to break the protein bonds of Taq polymerase, rendering the enzyme
unable to perform it’s function in the PCR(Kreader, C .A., 1996).

Considering the many problems which can be encountered prior to and during
amplification, concern has arisen over the validity of genetic typing results. False
positives fiom operator contamination have been traced to shed skin from exposed
facial areas. Appropriate use of protective lab clothing, the use of disposable gloves
and negative controls throughout the PCR process have minimized the problems of
Operator contamination(Kitchen, et al., 1990). Studies have shown that damage to the
DNA molecule by UV sources will generally stop the polymerase enzyme fiom acting

at the damaged site (Kobilinsky, L., 1992).

Chapter 3

Materials and Methods

Prior to the involvement of human subjects, approval was obtained through
the University Committee on Research Involving Human Subjects (UCRIHS).
After complete review of the project methods and goals, informed consent was
granted for one year. A total of 19 human subjects were used in the course of this
study to determine if shed skin cells could be obtained from bed sheets for
amplification by the polymerase chain reaction and accurate typing of six genetic
loci.

Subject Selection

Individuals were accepted for this study based on criteria that would
represent a variety of lifestyles. To avoid bias which might have had an effect on
the subject’s shed skin cell quantity or quality, no restraints were given to any
subject regarding sleep or nightly routine prior to the subject contact on the bed
sheets provided by the study. Subjects were chosen who reported sleeping
separately to enable clearer interpretation of the subjects genetic type which might
show secondary alleles from another individual in the same environment.

All subjects accepted into this study were ambulatory individuals who were

involved throughout the study with work, school or daycare. Subject ages ranged

12

13

from 4 through 80 years. Young subjects included were not diaper-dependent to
exclude the possibility of urinary cells on bedding. Teen and child participants had
their body weight noted to lend any insight in the event body weight or surface
area would appear to affect DNA quantity results. No subject was included who
would have a known, prolonged exposure to their bed sheets to bias DNA retrieval
quantity.

Collection of Cells

New, unwashed sheets were supplied to each of the 19 human participants
for shed skin cell retrieval. Exposure intervals to the bed sheets ranged from 10.5
to 143 hours. Each subject was designated numerically. Five mouth swabs were
collected for each subject for use as a standard for comparison to skin cell
specimens. The source of DNA specimen was designated “M” for mouth swab,
“B” for adhesive retrieval and “V” for vacuum retrieval. All adhesive specimens
were scraped randomly with a sterile blade and checked microscopically for the
presence of epithelial cells. Adhesives which did not Show epithelial cells were not
re-checked for cells due to any effect on data outcome, particularly when cellular
retrieval was anticipated to be decreased due to minimal exposure.

Subjects were instructed to place the new bed sheet set (one fitted sheet,
one flat sheet, one standard size pillow case) on their bed for a specified time
interval. A clean, bleached lab coat and disposable gloves were worn with each cell
retrieval. Skin cells were collected by the Adhesive Roller Method (Appendix A).

An adhesive strip was pulled from the roller after subject number 6 during the first

l4

adhesive retrieval for use as a negative control. Negative controls were also used
after subject’s numbered 6 and 16 during the second adhesive retrieval.

Subjects numbered 1 through 7, 12,13 and 16, were re-exposed to bed
sheets which had been washed and dried. Cells were collected by the Vacuum
Retrieval Procedure (Appendix B). Retrieval involved all surface areas of the
pillowcase, flat sheet and fitted sheet toward the subject side of the bed linen.
Vacuum filter retrievals were not checked for the presence of epithelial cells due to
the fragile nature of the filter paper and concern regarding the loss of subject skin
cells while manipulating the dried filter.

These subjects were then re-exposed to washed bed sheets at designated
intervals for a second adhesive retrieval. The presence of epithelial cells from the
second adhesive were observed microscopically. Exposure time was noted after
each cellular retrieval Age, floor contact, bathing, exercise habits, topical body
creams and household animals were noted initially for each subject to determine
comparison of these variables to quantities of cells retrieved by either method.
Specimen Transport

The skin cell specimens were collected for each subject then placed in a
separate envelope and labeled with the subject’s corresponding number and letter.
Subject cotton swabs from the inner cheek of the mouth were placed in a separate
paper envelope. Specimens were not refrigerated during transport.

DNA Extraction - Method I
The adhesive specimens were cut into small pieces approximately 5

millimeters by 10 millimeters. Adhesive or mouth swab Specimens were then

 

 

15

placed into their respective sterile 50 ml plastic tube. Five milliliters (ml) of Stain
Extraction Bufi‘er (Appendix H) was added to each tube to break epithelial cell
membranes, releasing the DNA and 100 ul Proteinase K to degrade proteins in
the reaction mixture. All tubes were vortexed and incubated at 56° C overnight.
Specimen DNA was extracted according to the Ethanol Precipitation Extraction
Method (Appendix C).
DNA Extraction - Method 11
Both the subject’s vacuum specimen fi'om the filter paper and the second
adhesive retrieval specimen were extracted by the Centricon® Extraction
Technique (Appendix D). In this method, the subject’s epithelial cells were
removed fiom the adhesive or vacuum filter surface by a series of washes with
18 ohm (millipore) water. It was not known at this time if the adhesive was
water soluble. After washing for cell removal, phenol/chloroform was used to
extract the DNA. Contents of the reaction mixture were placed in a Centricon®
100 filtration device for collection per manufacturers instructions.
BSA Addition
Prior to amplification, 5 microliters(ul) of bovine serum albumin were

added directly to the specimen tubes to enhance the action of Taq Polymerase to
bind with possible inhibitors of the PCR.
DNA Quantification

Quantitation of the DNA extracted from the skin cell specimens was
performed by Slot Blot (Appendix E). In this method, DNA is immobilized on

to a nylon membrane. A primate-specific probe (D17Z1) labeled with biotin is

l6

bound to the DNA for visualization. Addition of horseradish peroxidase-
streptavidin complex in the presence of a luminescence containing reagent results
in the emission of photons. The membrane is placed against radiographic film for
15 minutes exposure and developed by standard X-ray techniques. The intensity
of photon emission from the specimen is compared to known standards to
determine the quantity of DNA present in the sample in nanograms (ng) per
microliter (ul). Quantification of amounts as small as 0.15 ng can be detected by
this method but degradation of the DNA molecule can not be detemtined.
Mouth swab (buccal cell) DNA quantities were determined by Yield Gel
(Appendix F). This procedure quantifies 500 - 15 ng of DNA and can indicate
specimen degradation but is not primate-specific. Results were then calculated to
determine the respective amounts of DNA present for each mouth.
DNA Amplification and T wing

Mouth and skin cell specimens were amplified according to manufacturers
instructions for DQAl and Polymarker (Perkin Elmer®) for specimens
numbered one through seven at quantities of 0. lng/ul to 0.5 ng/ul in TE
Buffer(Appendix G). A post-amplification gel was performed and results
photographed.

Five adhesive specimens and corresponding mouth specimens were
re-amplified using increasing amounts of target DNA. Specimen 1B was
re-amplified at 12ng/20ul total TE Bufi’er(0.6ng/ul), 4B re-amplified at 9.8ng/20
ul total TE Buffer (0.49ng/ul) and 12B, 13B and 16B were re-amplified at

20ng/20ul total TE Bufi’er(1ng/ul) and results photographed.

17

Forty microliters(ul) of AmpliType® DQA1+PM reaction mix was placed
into each specimen tube. The reaction mix contains dATP, dGTP, dCTP and
dTTP which act as extenders or building blocks for the DNA target region to be
amplified. Extension is accomplished by elongating primers which flank the
variable regions of interest. The enzyme Taq Polymerase contained in the
buffered reaction mix acts as a catalyst for the reaction to proceed under
controlled conditions of temperature and time. Forty microliters (ul) of
AmpliType® PM Primer Set and 20 microliters(ul) of extracted sample were
added to each reaction tube and amplified for 32 cycles.

Genetic typing by reverse dot blot was performed on each specimen
amplified using the AmpliType® DQAl/ Polymarker kit for loci DQAl, LDLR,
GYP A, I-IBGG, D7 S8 and GC. All strips were first checked for the presence of
the control dot “C” which is the lightest color intensity and signifies valid test
system integrity of reagents, temperatures and time intervals throughout
amplification and genetic typing. The “C” dot is also used as an indicator of
color intensity when reading genetic type results. A color intensity equal to or
greater than the “C” dot is considered indicative of the specimen genetic result.

Eight DQAl alleles can be distinguished from each other in this test
method. There are three subtype designations for dot “l”: DQAl 1.1, DQAl 1.2
and DQAI 1.3. Dot number “2” designates the presence of DQAl allele 2 and
dot number “3” denotes the presence of DQAl allele 3. DQAI 4 has three
genetic subtypes which the test system denotes as DQAl 4.1 or DQAI 4.2/4.3

as the latter two alleles can not be distinguished by this test method.

18

As with the “C” dot on the DQAl test strips, the polymarker strips contain an
“S” dot which is used to compare intensities of the genetic type results. Neither
the “C” or “S” dot should appear on the negative control strip. Two different
alleles are possible for the LDLR and D788 variable regions and given the
designations “A” or “B”. The GYP A region has two alleles which can be
detected, although a third allele exists for this region. Thus, the designation for
GYP A is “A” or “B” also. There are three possible alleles at both chromosome
regions HBGG and GC. These are given designations of “A”, “B” and “C” for

the three genetic possibilities at these sites.

Chapter 4

Results

Part I - First Acfltesive Retrieval
Hours of Exposure and Cellular Quantities

Data from the subjects questionnaire is found in Figure 4 - 1, Figure 4 - 2 and
Figure 4 - 3. Figure 4 - 1 Shows subject exposure intervals which ranged from 143
hours to 80 hours. The average cellular quantity retrieved was 1 cell observed per
400X magnification field. Exposure intervals ranging fi'om 66 hours down to 10.5
hours also averaged 1 cell observed per microscopic field.

Subject A ge/Weight and Cellular Quantities

Subjects listed in Figure 4 - 1 which ranged in age from 47 through 80 years
averaged 2 cells observed per 400x magnification field. Ages 4 years through 17

years averaged 1 cell observed. Body weight of younger subjects was noted to
investigate possible effect of weight on cellular or DNA quantities. Subjects grouped
by body weight greater than 100 pounds produced an average of 1 cell seen
microscopically. No cells were seen from 2 subject’s in this group. Those weighing
less than 100 pounds also averaged 1 cell seen microscopically. No cells were

observed from 4 subject’s in this category.

19

20

Subject Floor Contact/Bathing/Exercise and Cellular Quantities

Figure 4 - 2 notes subject exposures to floor surface, bathing and exercise

prior to bed sheet exposure to determine any effect these variables might have on

cellular retrieval quantities.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SUBJECT HOURS SUBJECT AGE/ EPITHELIAL
NUMBER EXPOSURE WEIGHT CELLS
(POUNDS) (400X
MAGNIFIED)

l 80 50 0 - 2

2 66 80 0 - l

3 110 77 0 - l

4 143 I3 / 125 NONE SEEN
5 90 16 / 140 0 - l

6 102 17 / 170 0 - 1

7 48 9 / 60 O - 1

8 42 ll / 72 0 - l

9 45 16 / 140 NONE SEEN
10 56 47 0 - 2

11 52 48 0 - 2

12 110 8 / 51 NONE SEEN
13 110 5 / 35 0 - 2

l4 l8 8 / 80 NONE SEEN
15 21 15 / 150 0 - 2

16 ll 4 / 48 0 - l

17 10.5 10 / 64 0 - 1

18 12 5 / 42 NONE SEEN
19 12 8 / 72 NONE SEEN

 

 

 

 

 

Figure 4 - 1 Subject Data From First Adhesive Retrieval

Eleven subjects acknowledged contact to their floor environment prior to sleeping
on bed sheets distributed for study purposes. From these subjects, sixty-four
percent (7/11) showed epithelial cells microscopically from bed sheet retrieval.

Thirteen subjects noted bathing prior to bed sheet exposure. Of these subjects,

21

62% (8/13) were positive for the presence of epithelial cells. Fourteen subjects
expressed various forms of exercise or play activity prior to bed sheet exposure.

Sixty-four percent (9/14) who were active prior to bed sheet exposure showed

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

epithelial cells microscopically.
SUBJECT NUMBER FLOOR CONTACT BATHE PRIOR EXERCISE
PRIOR TO SLEEP TO SLEEP PRIOR TO SLEEP

1 YES YES YES
2 NO TWICE NO
3 NO ONCE NO
4 YES TWICE YES
5 YES YES YES
6 YES YES YES
7 YES YES YES
8 YES NO YES
9 NO NO NO
10 NO NO YES
1 1 NO NO YES
12 NO YES YES
13 NO YES YES
14 YES YES YES
15 YES YES YES
16 NO NO NO
17 YES NO NO
18 YES YES YES
19 YES YES YES

 

 

 

 

 

 

Figure 4 - 2 Subject Data From Questionnaire

Subject Additional Data From Questionnaire

Subjects involved in the study were questioned regarding any additional factors
which would lend insight in the event cellular or DNA yields would be decreased
(Figure 4 - 3). Three subjects used topical creams before bed sheet exposure.
Epithelial cells were observed for all three individuals. The presence of animals

living in the subjects homes was noted as some animals were known to have contact

with the subjects bed during exposure times. Animals which did not have contact

with subject bed sheets but were handled by the subjects prior to exposure intervals

were noted for each individual. Fourteen subjects had at least one animal in their

home during the course of this study. Of the fourteen who had animal contact, 57%

(8/14) showed epithelial cells microscopically.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SUBJECT NUMBER ADDITIONAL COMMENTS

1 NONE
2 USED TOPICAL STEROID ON NECK
3 USED MENTHOL CREAM ON BACK
4 1 DOG IN HOME
5 1 DOG IN HOME
6 2 DOGS IN HOME
7 1 DOG IN HOME
3 l DOG IN HOME
9 l DOG IN HOME

10 NONE

1 1 PSORIASIS CREAM USED ON LEGS

12 IDOG, 1 GUINEA PIG INHOME

13 IDOG, 1 GUINEA PIGINHOME

14 l DOG, 2 CATS IN HOME, RECENT HEAD

LICE INFECTION

15 lDOG,2CATSINHOME

16 IDOG,1FERRETINHOME

17 IDOG,IFERRETINHOME

13 l DOG IN HOME

19 1 DOG IN HOME

 

 

Figure 4 - 3 Subjects Additional Data

Comparison of Exposure Intervals to DNA Quantities

Figure 4 - 4, Figure 4 - 5 and Figure 4 - 6 show DNA yields of the 19 subjects by

Slot Blot Method. Standard DNA concentrations of lOng, Sng, 2.5ng, 1.25ng,

 

23

0.625ng, 0.3125ng and 0.15ng were used for subject quantity determinations.
Calibrators used as controls were as follows: Calibrator 1 (range 2.5 - 5.0ng),
Calibrator 2 (range 0.3125 - 0.625ng). An adhesive strip pulled after subject
number 6 (Figure 4 - 4) served as a negative control to investigate any subject-to-
subject contamination fiom the adhesive roller. Specimen 208 shows negative DNA
retrieval for bed sheets which did not come into contact with study subjects to
investigate DNA fiom the bed sheet manufacturer prior to subject exposure
intervals. Specimen 213 shows negative DNA yield for bed sheets which were
home laundered after subject exposure (Figure 4 - 6). This investigated the

possibility of DNA carry-over to subject’s re-exposed to laundered sheets during

the study.
IONG - - CAL l.=3.5NG NEG. CONT.
5 N6 _ __ CAI..2 =0.6NG
1.25 NG — - 28
0.62 NG ,__.. _ 33
0.312 NG ,,, - 43
0.15 NG
- 5B

Figure 4 - 4 First Group Bed Sheet DNA by Slot Blot

24

- - 133

10 No
5 NO . — 143
2.5 No '- -- 7B -- 153
1.25 NG - . -- 83
0.62 NG -- - 93
0.31NG .....,, .__~ 108
0.15 NG __ “B
'-' 12B

Figure 4 - 5 Second Group Bed Sheet DNA by Slot Blot

lONG - - CAL. l=2.5NG
5NG -- --- CAL2=0.31NG
Z-SNG — .— 168
1.25NG "' r73
062NG -- 188
I93
0.3ING -- 203
0.15NG 213

Figure 4 - 6 Third Group Bed Sheet DNA by Slot Blot

25

Subject exposure intervals which ranged from 80 to 143 hours averaged 106
hours exposure per subject with an average DNA yield of 6.8ng (Table 4 - 1).
Those with lower exposure intervals of 10.5 to 66 hours averaged 32.8 hours
exposure per subject and produced an average of 3.6ng DNA per subject.

Subject A ges/Weights Compared to DNA Quantities
Subjects whose ages ranged from 47 to 80 years averaged DNA quantities of 4.4ng
DNA for an average exposure time of 72.8 hours per subject (Table 4 - 1). Those
subjects who ranged from age 4 to 17 years averaged DNA quantities of 4. 12ng
with an exposure interval average of 55 hours per subject. DNA yield was not
detected on three subjects having exposure times of 12 hours ( 2 subjects) and 10.5
hours (1 subject). Subjects having weight noted below 100 pounds averaged
3.89ng DNA with exposure time averaged at 41.5 hours per subject. Body weights
recorded above 100 pounds averaged 4.6ng DNA with exposure times averaged at
80.2 hours per subject. Body weight of subjects not noted on the questionnaire
were not estimated or included in calculations.
Subject Epithelial Cells Observed Microscopical/y Compared to DNA Yield
Thirteen out of nineteen subjects showed epithelial cells microscopically. Of
those positive for the presence of skin cells, five subjects averaged 0 - 2 cells per
microscopic field. The DNA yield of these subjects averaged 2.06ng per subject
with an average exposure interval of 63.8 hours. Eight out of nineteen subjects
were positive for the presence of skin cells averaged 0 - 1 cells per microscopic

field.

Table 4 - 1 First Adhesive Retrieval DNA Quantities

26

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SUBJECT HOURS SUBJECT AGE/ EPITHELIAL DNA IN
NUMBER EXPOSURE TEEN OR CELLS SEEN NANOGRAMS

CHILD (400X

WEIGHTIN MAGNIFIED)

POUNDS
1 80 50 O - 2 2.5
2 66 8O 0 - 1 12.0
3 110 77 0 - 1 5.0
4 143 13 / 125 NONE SEEN 5.0
5 9O 16 / 140 O - 1 10.0
6 102 17 / 170 O - 1 5.0
7 48 9 / 60 0 - l 1.25
8 42 ll / 72 0 - 1 1.25
9 45 16 / 140 NONE SEEN 2.5
10 56 47 O - 2 1.25
11 52 48 O - 2 1.25
12 110 8 / 51 NONE SEEN 15.0
13 1 10 5 / 35 0 - 2 5.0
14 18 8 / 80 NONE SEEN 2.5
15 21 15 / 150 O -2 0.3
16 11 4 / 48 0-1 10.0
17 10.5 10 / 64 0-1 0
18 12 5 / 42 NONE SEEN 0
19 12 8 / 72 NONE SEEN 0

 

These eight subjects averaged a DNA yield of 5.6ng per subject with average

exposure times of 59.5 hours. Subjects numbered 4, 9, and 12 who did not Show

skin cells microscopically yielded DNA quantities of 5.0ng, 2.5ng, 15.0ng and 2.5ng

respectively. Subject number 17 averaged 0 - 1 cells microscopically and was

negative for DNA yield.

 

27

Amplification of First Acflresive Retrieval Specimens

Figure 4 — 7 and Figure 4 - 8 show amplification results of subject’s numbered
1 through 7 for mouth specimens and first adhesive retrieval specimens extracted by
the Ethanol Precipitation Method. Amplification was performed in a Perkin-Elmer®
PCR thermal cycler (model 480) for all six genetic loci simultaneously.
Amplification success was verified by GIBCO BRL® 123 base pair ladder with
ethidium bromide for visualization of six bands. The post-amplification gel showed
inhibition of all 7 adhesive retrieval specimens with no indication of inhibition for

the corresponding subject mouth specimen.

 

Figure 4-7 Subjects 1,2,4 and 5 Mouth and Bed Sheet DNA Amplifications

28

LADDER

3M

 

Figure 4-8 Subjects 3, 6 and 7 Mouth and Bed Sheet DNA Amplification

Re-amplified DNA From F irst Adhesive Retrieval

To investigate severe degradation of the subjects DNA which might have an
effect on target sequences during amplification, subject’s numbered 1, 4, 12, 13 and
16 were re—amplified at increased DNA concentrations. Figure 4 - 9 shows
post-amplification results of subject number 4, re-amplified at an increased total
concentration of 9.8ng DNA . Figure 4 - 10 shows post-amplification of subject
number 1 at 12ng and subjects numbered 12, 13 and 16 at increased total DNA
concentrations of 20ng. All re—amplifications used the corresponding subject’s
mouth specimen at the original dilution and GIBCO BRL® 123 base pair ladder for
verification of test system integrity. All mouth specimens amplified successfully.
Adhesive specimens at increased concentrations showed no evidence of successful

amplification.

29

 

Figure 4 - 9 Subject 4 Re-Amplified DNA from Adhesive Retrieval

LADDER

1M

18 AT 12 NO
12M

lZB AT 20 NO
13M

13B AT 20 NO
16M

168 AT 20 NO

 

Figure 4-10 Subjects 1,12,13,16 Re-Amplified DNA from Adhesive Retrieval

Part II - Vacuum and Second Adhesive Retrieval by C entricon® 100 Extraction
Figure 4 - l 1 shows DNA quantities by Slot Blot for subject’s 1 through 7, 12,
13 and 16 who were re-exposed to washed bed sheets for vacuum retrieval. These

same subject’s were the exposed once more to washed bed sheets for a second

‘1 l“ I~

.‘v'«-

‘0!”‘.

30

adhesive retrieval. Both the vacuum retrieval and second adhesive retrieval
specimens were then extracted by the Centricon® 100 Extraction technique.
Quantities of DNA were compared to known standard DNA quantities of 10ng,
Sng, 2.5ng, 1.25ng, 0.62ng, 0.31ng and 0.15ng with Calibrator 1 (range 2.5 - 5.0ng
DNA) and Calibrator 2 (range 0.3125 - 0.625ng DNA) as test system controls.
Negative control 1, adhesive pulled from the roller after subject number 6 and

negative control 2, adhesive pulled afier the last subject numbered 16 both

produced negative DNA yields.
CAL 1
011.2
5 N0 - ..- - 12V .- 1.
LING - .. ‘v an. 13V '. ‘23
1.25m ‘- 1V --- "V :B .- us
car no -- -- 3V 33 ten
MING .. ..... 4V 48 meow“
9 Into .. _ 5V as Neocorrrz
6V 63

Figure 4 - 11 Slot Blot of Vacuum Retrieval and Second Adhesive Extraction

Table 4 - 2 shows quantities of DNA from the vacuum retrieval compared to

subject exposure intervals and subject age. Subject’s 2, 4, 5, 7 and 16 whose

31

average exposure interval was 45.7 hours, produced an average of 1.24ng DNA per
individual. Subject’s 1, 3, 6, 12 and 13 who averaged 67.8 hours exposure per
subject, produced an average of 1.69ng DNA per subject. Those subject’s whose
ages ranged from 17 to 80 years, averaged DNA quantities of 1.66ng DNA with an
average exposure interval of 62.6 hours. Subject’s whose ages ranged from 4 to 13

years averaged 1.27ng DNA with an average exposure interval of 50.9 hours.

Table 4 - 2 DNA Quantities from Vacuum Retrieval Procedure

SUBJECT NUMBER HOURS EXPOSURE SUBJECT AGE DNA IN
NANOGRAMS

56 50 0.15
52.5 80 0.15
91 77 2.5
49.5 13 0.6

53.5 16 5.0
60 17 0.5
45 9 <0.15

66 8 5.0
66 5 0.3
28 4 0.3

 

Table 4 - 3 lists quantities of subject epithelial cells and DNA fi'om the second
adhesive retrieval with DNA extraction using the Centricon® 100 technique. The 5
oldest subject’s whose ages ranged from 16 to 80 years produced an average of 2
cells observed microscopically with an average exposure interval of 50.4 hours per
each subject in this age group. The 5 youngest subject’s in age range 4 through 13
years showed an average of 1 cell per subject with the average exposure interval of

56.5 hours per subject. Adhesive pulled afier subject’s numbered 5 and 16 are

32

shown as negative controls. Exposure hours, subject age and epithelial cells are not

compared to DNA quantities for this group as DNA yield was affected by

insolubility of the adhesive with the Centricon® 100 extraction technique.

Table 4 - 3 DNA Quantities From Second Adhesive Retrieval

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SUBJECT HOURS SUBJECT AGE EPITHELIAL DNA IN
NUMBER EXPOSURE CELLS SEEN NANOGRAMS
(400x
MAGNIFIED)
1 4s 50 0 - 2 NEGATIVE
2 48 so 0 - 1 NEGATIVE
3 42 77 o - 2 NEGATIVE
4 42 13 1 - 2 NEGATIVE
5 72 16 2 - 3 NEGATIVE
NEG. XXXXXXXXX )oooooooor XXXXXXXXX NEGATIVE
CONTROL
6 42 17 o - l NEGATIVE
7 59.5 9 o - 1 NEGATIVE
12 6o 8 o - 1 0.3
13 60 5 o - 2 NEGATIVE
16 63 4 o - l 0.6
NEG. XXXXXXXXX xxxxxrooor Iorxxxxxror NEGATIVE
CONTROL

 

Compared DNA Quantities of First Acflresive Retrieval to Vacuum Retrieval

Table 4 - 4 shows an average of 59.9 hours for the 19 subject’s collected by

adhesive retrieval with precipitation of DNA by ethanol. The average DNA quantity

extracted from these subject’s was 4.2ng per subject. Ten of these subject’s,

collected by vacuum retrieval with Centricon® 100 extraction, have an average

 

33

exposure of 56.8 hours per subject and produced an average DNA yield of 1.47ng

per subject.

Table 4 - 4 Compared DNA Quantities - Vacuum Retrieval to Adhesive Retrieval

 

Amplification of Vacuum Retrieval Specimens

Figure 4 - 12 and Figure 4 - 13 show amplification of the 10 subject’s DNA
retrieved by vacuum technique. Corresponding mouth swab specimens were
amplified for each of the subject’s. Successful amplification of desired PCR products

was verified by the presence of 6 bands in the123 base pair GIBCO® BRL ladder.

a
.III N
‘.

 

.1

Figure 4 - 12 Mouth and Vacuum Amplification of Subjects 1-5

 

Figure 4 - l3 Mouth and Vacuum Amplification of Subjects 6, 7, 12, 13 and 16

Amplification of Second Adhesive Retrieval Specimens
Figure 4 - 14 illustrates amplification of the 2 second adhesive retrieval
specimens successfirlly extracted by Centricon® 100 technique. The negative
control used was pulled from the adhesive roller between subject’s numbered 5 and

6 during the second adhesive retrieval.

LADDER
123

168

NEG. CONT.
POS. CONT.

 

Figure 4 - 14 Second Adhesive Amplification of Subjects 12 and 16

Io‘

.-’Y"mr4

‘OOAI

35

Results of Genetic Typing

Figure 4 - 15 and Figure 4 - 16 show genetic types of the 10 subject’s
amplified for DQAl alleles using Centricon® 100 Extraction. Subject’s were given
the same numerical designation on the test strip as assigned during the study.
Specimen sources were designated as M = Subject Mouth Swab, V = Subject
Vacuum Retrieval Specimen and B = Subject Second Adhesive Retrieval Specimen.
All test strips were first verified for presence of the control dot (“C” dot).
Intensities greater than or equal to the control dot were noted as the subject genetic
type. Sequence specific probes used for amplification of the particular genetic
regions of interest are human-specific and will not detect DNA from other sources

such as household animals.

.0. . .9; .. .3 n

ll
0
N rt
0
I

.. '1 in“
u ‘

III

jam 7 \l

"m \I I“

~- ‘1‘,

III :h :l: :l: T." ;I: u: .h .1! .n .1: ;u :l: :1: ill 5

O
.

e
n =3 H r: .. :

 

Figure 4 - 15 Subjects 1-7 and 12, Mouth and Vacuum Retrieval DQAl Results

in

‘I‘IN

4'
0

F132

'7‘

153‘.

I
J’,

4

3351032:

.\ ‘.
~ I.

:4

s .

M.

‘
t
h 1o
I
..
.
.
0 9n

\a)

4-,

36

uuuuuuu

uuuuuuu

 

Figure 4 - 16 Subjects12,13 and 16 Mouth, Vacuum and Adhesive DQAl Results

Figure 4 - 17 shows the interpretation of the subject’s DQAl genetic types
as compared to the control dot intensity included on each subject’s test strip.
Subject’s numbered 1 V (vacuum retrieval) and 16 V (vacuum retrieval) were
negative for genetic type results. DNA quantities of 0. 1 5ng (Subject 1) and 0.3ng
(Subject 16) from Slot Blot quantification (Figure 4 - 11) were detected. The post-
amplification gels (Figure 4 - 12 and Figure 4 - 13) of both specimens for the six
genetic loci of interest appeared faint. The adhesive strip used as a negative control
throughout the test procedure was negative for all genetic types. The positive
control genetic result of HLA DQAI 1.1, 4.1 correlated to AmpliType®
performance characteristics per manufacturers instructions. Figure 4 - 18 and Figure
4 - 19 show genetic types of the subject’s polymarker loci (LDLR, GYP A,
HBGG, D788 and GC). Interpretation of each subject was based on intensity of
each test area compared to a control dot on the subject’s test strip ( “S” dot).
Intensities greater than or equal to the “S” dot were considered positive for the

subject’s genetic type.

37

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SUBJECT DQAI TYPE
NUMBER
1M 1.1, 4.2/43
1V NORESULT
2M 1.2, 4.1
2v 1.2, 4.1
3M 1.2, 3
3v 1.2, 3
4M 1.1, 1.1
W 1.1, 1.1
5M 2, 4.1
SV 2, 4.1
6M 1.3, 4.1
6v 1.3, 4.1
7M 1.3, 1.3
7v 1.3, 1.3
12M 1.1, 1.1
12V 1.1, 1.1
13M 1.1, 1.1
13V 1.1, 1.1
16M 1.1, 4.2/4.3
16v NO RESULT
12B 1.1, 1.1
CENTRICON®
16B 1.1, 4.2/4.3
CENTRICON®
NEGATIVE NEGATIVE
CONTROL
POSITIVE 1.1, 4.1
CONTROL

 

Figure 4 - 17 Interpretation of DQAl Results Vacuum and Adhesive

38

 

Figure 4 - 18 Subjects 1-6 Mouth and Vacuum Polymarker Results

 

Figure 4 - 19 Subjects 7, 12, 13 and 16 Polymarker Results

39

Interpretation of the subject’s polymarker results are given in Figure 4 - 20 for both
vacuum and second adhesive retrieval specimens. Subject number IV was again negative
for genetic loci type. All subject’s Showed correlation of mouth swab specimens used as a

comparison standard to vacuum retrieval specimens.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SUBJECT LDLR TYPE GYP A TYPE HBGG D788 GC TYPE

NUMBER TYPE TYPE
1M AA AB AA AA AA
1V NO RESULT NO RESULT N0 RESULT NO RESULT NO RESULT
2M AB AB AB AB AB
2V AB AB AB AB AB
3M AB AB AA AA CC
3v AB AB AA AA CC
4M AB AB BB AA CC
4v AB AB BB AA CC
5M BB AA AB AA CC
5v BB AA AB AA CC
6M AB AB AB AB AC
6v AB AB AB AB AC
7M AB AB BB AB CC
7v AB AB BB AB CC
12M BB AB AB BB BB
12v BB AB AB BB BB
13M AA BB AB BB BC
13V AA BB AB BB BC
16M AB AA BB AA CC
15v AB AA BB AA CC

12B BB AB AB BB BB

CENTRICON®

168 AB BB AA CC

CENTRICON®

NEG. NEG. NEG. NEG. NEG. NEG.

CONTROL

POSITIVE BB AB AA AB BB

CONTROL

 

Figure 4 - 20 Interpretation of Polymarker Results Vacuum and Adhesive

 

40

Subject’s numbered 12 and 16 showed comparison of mouth specimens to both
vacuum retrieval and second adhesive retrieval specimens. The negative control used
throughout the study showed no sign of any genetic alleles. The positive control supplied
from the test manufacturer corresponded to expected results given in the AmpliType® user
guide.

Part III - Subject Mouth Swab Specimens

Figure 4 - 21 and Figure 4 - 22 show results from subject mouth swab specimens
used as standards for comparison to the vacuum and adhesive results. All 5 mouth swabs
collected fi'om each subject were extracted by the Ethanol Precipitation Method and
quantified by agarose gel electrophoresis with ethidium bromide used for visualization. All
mouth swab quantities were compared to known standard DNA concentrations. Specimen
intensities were compared to the standard concentration intensities for SOOng, 250ng,

125ng, 63ng, 31ng and 15ng to estimate total DNA quantity. Each subject’s total

 

Figure 4 - 21 Subjects 1 through 6 Mouth Swab DNA

' .. ". -‘,_
4.0
v
‘4‘ 0,... ‘.
O
. - .L‘L.‘ 1;.
. , _

T '0‘.“ .

“>-.

 

41

 

Figure 4 - 22 Subjects 7 - 19 Mouth Swab DNA

DNA from the 5 mouth swab’s was diluted to a final concentration of 0.1 - 0.5ng/ul
in TE Bufi‘er for amplification. Table 4 - 5 shows total mouth swab quantities

determined for each subject as compared to DNA standard concentrations.

Table 4 - 5 Subjects Mouth DNA Quantities

DNA

 

Chapter 5

Conclusions and Recommendations

This study evaluated the potential for using shed skin cells as a source of
DNA for comparison purposes to aid in the identification of missing persons.
Variables affecting cellular and DNA quantities were also studied including two
retrieval and two extraction methods. It was found that shed skin cells are a very
rich source of DNA which can be used as a standard for comparison to another
source of DNA fi'om the human body. One variable, subject age, was determined
to afi’ect the quantity of cellular retrieval seen microscopically. Older subjects in
age range fiom 47 through 80 years, were found to yield twice the amount of shed
skin cells than younger subjects in age range 4 through 17 years. DNA quantities
for these same age ranges were found to be unafi’ected. Correlation of cellular to
DNA quantities may be found if the entire adhesive areas could have been viewed
which was not able to be accomplished in this study.

DNA quantities were affected by subject exposure intervals only, with
longer exposures producing greater DNA quantities. No other variables
investigated in the study affected DNA quantities. Table 5 - 1 shows cellular and
DNA yields fiom subjects who noted floor contact, bathing, activity, body creams

and animal contact during exposure intervals. Variables listed in Table 5 - 1 did not

42

43

impact cellular or DNA findings. The percentage of subjects yielding DNA who

listed floor contact, activity and household animals also included 3 subjects who

had lower exposure intervals (subject 17 - 10.5 hours, subject 18 - 12 hours and

subject 19 - 12 hours). The lower exposure intervals would have an effect on data

outcome in this group.

Table 5 - 1 Percent of Subjects Yielding Cells and DNA

 

PERCENT SUBJECTS

PERCENT SUBJECTS

 

 

 

 

 

 

 

 

YIELDING CELLS YIELDING DNA
FLOOR CONTACT 62 76.9
BATHING 62 100
ACTIVITY 64 78.6
TOPICAL CREAMS 100 100
ANIMAL CONTACT 57 78.5

 

The Adhesive Retrieval Method was found to be significantly more

efficient than the Vacuum Retrieval Method. Table 4 - 4 shows an average

exposure interval of 56.8 hours for vacuum retrieval subjects. This is 5% lower

than adhesive retrieval subjects averaged at 59.9 hours exposure. The average

quantity of DNA retrieved from the vacuum subjects was 35 % lower than those

retrieved by adhesive. The adhesive strip had the advantage of concentrating the

DNA source in a smaller area and was not susceptible to loss of the shed skin cells

within the internal environment of the vacuum components. Additionally, the

adhesive roller was lightweight, rapid to use and economical without additional

consideration for power source or decontamination measures.

 

44

Figure 5 - 1 shows the order in which vacuum retrieval subjects were
collected and the secondary alleles (lighter intensities than control dot on dot blot
strip) which appeared. The presence of secondary alleles in the first subject

retrieved (subject number 3), and the absence of secondary alleles for subject’s

 

 

 

 

 

 

 

 

 

 

 

SUBJECT DQAl LDLR GYP A HBGG D7S8 GC SECONDARY
NUMBER ALLELES
3 12, 3 AB AB AA AA CC 33.44.1113“?
0788 B
GC AB
5 2, 4.1 BB AA AB AA CC NONE
7 1.3, 1.3 AB AB BB AB CC 2.3.4.1
HBGG A
GC AB
2 1.2.4.1 AB AB AB AB AB 2.3.113ch
CC C
6 1.3, 4.1 AB AB AB AB AC 2,GC a
12 1.1, 1.1 BB AB AB BB BB NONE
13 1.1, 1.1 AA BB AB BB BC 4.1,GYPA A
4 1.1, 1.1 AB AB BB AA cc 1.2. 2. 3. 4.1
1 NO NO NO NO NO NO RESULT NO
RESULT RESULT RESULT RESULT RESULT RESULT
16 NO RESULT AB AA BB AA CC OYPA B
HBGG A
0788 B
GC AB

 

 

 

 

 

 

 

 

 

 

Figure 5 - 1 Order Of Vacuum Specimens Showing Secondary Alleles

numbered 5 and 12, could indicate a random carry-over between subject’s

retrieved during collection and may not have come from the subjects external

45

environment. Figure 5 - 2 shows the 2 subject’s amplified by the second adhesive

retrieval. One subject showed the presence of a secondary allele (DQAI 4.1 only).

 

 

 

 

 

 

 

 

 

 

’ SUBJECT DQAI LDLR GYP A HBGG D788 Gc SECONDARY I
NUMBER ALLELES

|| 12 11,11 BB AB AB BB BB 4.1 I

| 16 1.1,4.2/4.3 AB AA BB AA CC NONE l

 

Figure 5 - 2 Order of Second Adhesive Specimens With Secondary Alleles

Subject environment can not be ruled out as no evidence of subject-to-subject

carryover was found by Slot blot for negative control adhesive strips (Figure 4 - 4

and Figure 4 - 11) during adhesive retrieval.

The Centricon® 100 Extraction Method with BSA addition produced

DNA suitable for amplification. Quantities of DNA extracted from the second

adhesive retrieval were affected by insolubility of the adhesive in water used

initially to remove the cells fi'om the adhesive. The Ethanol Extraction Method

was effective in extracting DNA fi'om the adhesive medium but was ineffective for

separating amplification inhibitors present with the extracted DNA. To remove all

cells fiom the adhesive medium and separate amplification inhibitors, organic

extraction with Centricon® 100 concentration could produce sufficient DNA

quantities fi'om adhesive media which would be separated fiom inhibitors. The

DNA would then be suitable for amplification with the addition of BSA.

Additional studies should be performed to investigate the potential for shed

skin cells as a standard for comparison in forensic investigations. Utilization of

46

more discerning PCR techniques, such as STR determinations could differentiate a
mixture of genetic types, consisting of more than one individual, as opposed to the
sequence Specific polymorphism’s used in this study which are less discriminating.

The subject’s involved throughout this study were exposed to their bed
sheet’s during restful periods during times the subject normally slept. Greater shed
Skin cell quantities may be retrieved from a subject’s environment afier periods of
stressfirl exposure such as physical struggle. Shed skin cells retrieved with adhesive
for subsequent DNA genetic typing may be usefirl in forensic investigations to
establish the presence of an individual or group of individuals to a particular
environment.

A pre-packaged, sterile, adhesive roller with disposable handle should be
considered for use during criminal investigations involving cellular retrieval to
avoid issues involving carry-over from one subject environment to another. Using
an adhesive retrieval method, large areas of a suspect environment can be screened
for the presence of DNA sources from a variety of surface media, such as carpet,
bedding and other items found in homes and automobiles. Suspect clothing may
also be screened by adhesive retrieval for DNA sources. This study has shown that
shed skin cells are a rich source of DNA when concentrated on an adhesive
medium. The use of shed skin cells, easily recovered with adhesive, may have great
potential in assisting investigators in the identification of missing or unidentified

persons.

APPENDIX A

APPENDIX A

ADHESIVE ROLLER METHOD

Note: It is important to use Universal Precaution Techniques in the collection and

handling of any biological fluid or tissue to control any cross contamination between

the subject and the evidence technician. Therefore, the use of disposable gloves
throughout the entire collection procedure is necessary for proper retrieval.

1. Use a standard household adhesive roller with adhesive on one side only. Size of
the adhesive used in this study was 14 centimeters by 10 centimeters.

2. Pull one to two adhesive strips ofi’ prior to rolling bed sheets to avoid any
previous exposure to the environment.

3. Starting with the pillowcase (if used by the subject), roll the outer area of the
pillowcase in a slow backward and forward manner. Include the sides and edges
of the pillowcase.

4. Roll the fitted (bottom) sheet, Starting at the head or top of the sheet. It is
sometimes easier to roll the sheet width-wise standing over the bed to ensure that
all possible evidence from the fitted sheet surface will be retrieved.

5. Retrieve cellular material from the top sheet last as evidence from this sheet is

thought to be minimal. If the top sheet is not on the subject’s bed, do not roll this

47

4 8
sheet with the adhesive as issues of cross contamination fiom other family
members may be largely increased.

6. Place the subject’s bedding in a bag with the subject’s identification on the outside
of the bag. Fold the bedding in a manner so that the exposed side of the bed sheets
does not come in contact with the external environment.

7. Place the subject’s adhesive in a new paper envelope with the subject’s

information on the envelope. Seal the envelope.

APPENDIX B

APPENDIX B

VACUUM RETRIEVAL PROCEDURE

Note: It is important to use Universal Precaution Techniques in the collection and

handling of any biological fluid or tissue to control any cross contamination between

the subject and the evidence technician. Therefore, the use of disposable gloves

throughout the entire collection procedure is necessary for proper retrieval.

1.

Extreme care must be exercised in cleaning the internal components of a hand
vacuum used for collection of cellular material. Coating of internal rough surfaces
such as the internal filter was accomplished by the use of foil. The foil was
changed with each new subject to avoid cross contamination. The chute of the
vacuum containing the opening to the outside environment was soaked with 70%
ethanol then rinsed with distilled water and dried thoroughly before each use.
Using a fully charged hand vacuum, line rough surfaces with new foil. Lightly
Spray a piece of Whatman filter paper using sterile, distilled water. The dimensions
of the filter paper used in this study were approximately 15 centimeters by

11 centimeters.

Attach the chute of the vacuum.

Vacuum the subject’s bedding Starting with the outer pillowcase in a slow
backward and forward manner, taking care to hold the tip of the vacuum in an
upward position vertical to the pillowcase. Include the sides and the edges of the

pillowcase.

49

50

5. Vacuum the fitted (bottom) sheet, starting at the head or top of the sheet,

ensuring that all possible cellular material will be retrieved.

6. Retrieve cellular material from the top sheet last due to suspected minimal
presence of evidence. Do not vacuum the top sheet if found on subject’s floor due
to possible cross contamination from other family members.

7. Place the subject’s bedding in a bag with the subject’s identification on the outside
of the bag. Fold the bedding in a manner so that the exposed side of the bed sheets
does not come in contact with the external environment.

8. Place the subject’s filter paper specimen in a paper envelope with the subject’s
information on the outside of the envelope. It is important to fold the filter paper
inward so the conical Shape of the internal vacuum filter is maintained, as most
cells will aggregate between the conical internal filter and the moistened filter
paper. This will ensure loss of cellular material will be minimal once the filter
paper dries completely inside the paper envelope until it is analyzed.

9. Swab the inside of the vacuum chute around the opening with a cotton swab
moistened with sterile, distilled water to collect cells which may have collected on
the inside of the vacuum chute and not on the filter paper itself.

10. Place subject’s moistened cotton swab in the envelope containing the filter paper.

APPENDIX C

APPENDIX C

ETHANOL PRECIPITATION EXTRACTION METHOD

Mouth Swab Specimen:

1.

2.

7.

8.

Cut tips ofl’ cotton swabs and place in 5 milliliters (ml) of Stain Extraction Bufi’er.
Add 100 microliters (ul) of Proteinase K.
Vortex 2 seconds.

Incubate overnight at 56° centigrade.

. Place cotton swab tips in spin-basket and centrifuge at 2000 rpm’s for 5 minutes.

Remove spin-basket and bring supernatant down to a volume of 410 111. DO NOT
DISTURB CELL PELLET.
Place the 410 III supematant/cell pellet into a sterile 1.5 ml capped tube.

Proceed with ethanol extraction protocol.

Bed Sheet Adhesive Specimen:

1.

Randomly scrape approximately 5 areas of adhesive with sterile scalpel and place
scrapings in distilled water. Place 1 drop of water mixture under low power light
microscope and observe for presence of epithelial cells.

Cut adhesive into small 1 centimeter (cm) by 2 cm pieces and place into two,

50 ml tubes. Add 5 ml Stain Extraction Buffer to each tube.

Add 100 III Proteinase K to each tube.

Vortex 2 seconds.

51

8.

9.

5 2
Incubate overnight at 56° centigrade.
Place adhesive cuttings in spin-basket and centrifirge at 2000 rpm’s for 5 minutes.
Remove Spin-basket and decrease supernatant to a volume of 410 111 in each tube.
Place 410 111 supematant/cell pellet into sterile 1.5 ml capped tube.

Proceed with ethanol extraction protocol.

Ethanol Extraction Protocol:

1.

In laminar flow hood, place 500 ul phenol/chloroform/isoamyl into each tube.
(Pipet from bottom). Vortex 2 seconds.

Microfuge 2 minutes at 10,000 rpm’s.

Transfer aqueous (top) layer into a new 1.5 ml sterile tube. Avoid removing
denatured protein that collects at the interface of the supernatant!

Add 1.0 ml Cold Absolute Ethanol.

Vortex 2 seconds and place the tube a -20° centigrade for 30 minutes.
Microfirge 15 minutes at 10,000 rpm’s.

Pour ofi’ alcohol (top layer).

Speed-Vac sample for 8-10 minutes to remove remaining ethanol.

Resolubilize the DNA in 36 ul TE buffer. Sample is now ready for quantification.

Store all samples at 2 - 6° centigrade until ready to use.

APPENDIX D

APPENDIX D

CENTRICON® EXTRACTION TECHNIQUE

1. Roll filter paper or cut adhesive sample and place in 50 ml tube with approximately
35 ml 18 ohm, distilled water. Vortex 30 seconds.

2. Place filter paper particles or cut adhesive pieces into a straining device. Centrifuge
for 3 minutes at 5000 rpms. Remove straining device and remove supernatant,
leaving approximately 10 ml of solution in tube bottom each time. Repeat if
necessary to remove all particles.

3. Wash two more times with 18 ohm, distilled water, centrifuging for 3 minutes each
time at 5000 rpms.

4. Remove supernatant to within 1.5 ml of cell pellet after the third wash and place

into a 2 ml plastic, capped tube. Spin this tube for 4 minutes at 14,000 rpms
and decant supernatant.

5. Wash the original (50 ml) tube twice again with 18 ohm, distilled water, decanting
the supernatant and adding the cell pellet from the original tube into the 2 ml
plastic tube.

6. Wash the original tube last with Tris-EDTA buffer and add cell pellet with buffer
for a final total volume of approximately 400 111 (cell pellet/ Tris-EDTA buffer).

7. To this tube, add 400 ul Stain Extraction Buffer and 10 ul Proteinase K enzyme.

8. Incubate tube overnight at 56° C.

53

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

54

Remove tubes from overnight incubation and spin 4 - 5 seconds at 14,000 rpms to
settle any condensation on tube sides.

Place 500 ul of Phenol/ Chloroform/ Isoamyl alcohol into each tube. (Pipet
from bottom to aspirate phenol portion). Shake well until “milky” appearing.
Centrifuge 8 minutes at 14,000 rpms.

Label Centricon® membrane support, sample reservoir and retentate vial.

Place 3 - 4 drops of Tris-EDTA Bufi’er on filter of membrane support - do not
touch filter.

Pipet aqueous (top) layer fi'om plastic, capped tube onto Centricon® filter.

Add 400 111 of Tris-EDTA Bufi‘er to plastic, capped tube. Shake until “milky” and
centrifuge tube again for 8 minutes at 14,000 rpms.

Place aqueous (top) layer fiom plastic, capped tube onto Centricon® filter.

Add Tris-EDTA Buffer to within 1 centimeter from the top of the membrane
support tube and centrifirge at 2,500 rpms for 30 minutes. Retain wash pellet.
Fill the membrane support tube again with Tris—EDTA Bufi’er and centrifuge for
30 minutes at 2,500 rpms. Retain wash pellet. Repeat wash again. Retain wash
pellet.

Fill membrane support tube with Tris-EDTA Buffer and centrifuge for 60
minutes at 2,500 rpms.

Place retentate vial on the Centricon® tube, invert and spin for 8 minutes at
3,000 rpms.

Determine the total amount of final volume in the with a calibrated pipet for slot

blot calculations.

APPENDIX E

APPENDIX E

SLOT BLOT PROCEDURE FOR SKIN CELL QUANTITATION

Note: DNA Standard A, DNA Calibrators (1 and 2), QuantiBlot® Probe (D1721),

Enzyme Conjugate (HRP-SA)and Bromothymol Blue Solution is supplied by

Perkin Elmer QuantiBlot® Human DNA Quantitation Kit.

1. Prepare serial dilution of the Standard A (10ng/5ul): Label tubes for dilution

A through G. Vortex the DNA Standard A to mix thoroughly. Transfer120 ul of
DNA Standard A into the tube labeled A. Aliquot 60 ul of TE Bufl‘er into each of
the six remaining tubes labeled B through G. Add 60 ul of DNA Standard A to the
60 ul of TE Bufi’er in tube B. Vortex to mix thoroughly. Add 60 ul of diluted DNA
Standard B to the 60 ul of TE Bufi’er in tube C. Vortex to mix thoroughly.
Continue the serial dilution through tube G.

2. Determine the number of samples to be analyzed including the seven Human DNA
Standards (A through G), the DNA Calibrators l and 2, and the one blank PCR
reaction tube consisting of spot solution only. Aliquot 150 III of Spotting Solution
into a new tube.

3. Label seven of the tubes containing spotting solution A through G and label two
of the tubes 1 and 2.

4. Vortex the seven DNA standards and calibrators and add 5 ul of each sample to
the corresponding labeled tube.

5. Add 5 ul of each test sample DNA to the other tubes containing 150 111 of

Spotting Solution.

55

10.

11.

12.

13.

56

. Cut a piece of Biodyne B membrane to fit slot blot apparatus. Cut ofl’ one corner

for orientation purposes.

Place the Biodyne B membrane into 50 ml of Pre-Wetting Solution for one to
thirty minutes.

Using forceps, remove the membrane from the Pre-Wetting Solution. Place the
membrane on the gasket of the slot blot apparatus, the place the top plate of the
slot blot apparatus on top of the membrane.

Turn on the vacuum source.

Turn ofi’ the sample vacuum and turn on the clamp vacuum. Push down on the top
plate to ensure a tight seal.

Pipet each sample into a difi’erent pre-deterrnined well onto the Slot blot. Dispense
each sample directly into the center of each well.

After all samples have been pipeted into the wells of the apparatus, slowly turn on
sample vacuum. Leave vacuum on until blue color appears at bottom of well.
Turn ofi’ sample vacuum, clamp vacuum and vacuum source.

Transfer the membrane to 100 ml of pre-warmed Hybridization Solution. Add 5
ml of 30% hydrogen peroxide. Rotate in 50° C water bath for 15 minutes. Pour

off the solution.

14. Add 30 ml of Hybridization Solution to the tray containing the membrane. Tilt

the tray to one side and add 20 ul of QuantiBlot D17Z1 Probe to the
Hybridization Solution. Place lid on tray and rotate in 50°C water bath for 20

minutes. Pour off the solution.

15.

16.

17.

18.

19.

20.

21.

57

Rinse the membrane briefly in 100 ml of pre-warmed Wash Solution by rocking
for several seconds. Pour off the solution.

Add 30 ml of the pre-warmed Wash Solution to the Hybridization tray. Tilt the
tray to one side and add the Enzyme ConjugateHRP-SA to the 30 ml of Wash
Solution. For chemiluminescent detection, add 90 ul of Enzyme Conjugate:
HRP-SA. Place the lid on the tray and rotate in a 50°C water bath for 10 minutes.
Pour 011’ the solution.

Rinse the membrane for 1 minute in 100 ml of pre-warmed Wash Solution by
rocking at room temperature on an orbital shaker for 15 minutes. Pour 011’ the
solution.

Rinse the membrane briefly inl 00 ml of Citrate Buffer by rocking the tray. Pour
Off the solution.

To 5 ml of ECL Reagent 2, add 5 ml of ECL Reagent 1. Prepare this reagent 5
minutes before use. Add the 10 ml of ECL reagent mixture to the membrane.
Shake the tray for exactly 1 minute at room temperature.

Expose the membrane to X-ray film within 10 minutes of ECL reagent incubation.

Exposure time may be 15 minutes to 2 hours for effective visualization.

APPENDIX F

APPENDIX F

YIELD GEL FOR BUCCAL CELL QUANTITATIVE ANALYSIS

. Weigh flask prior to use. Weigh 2.8 grams of Sigma Type H agar and combine
into 280 ml TAE Buffer. Weigh flask with agar and TAE Bufi’er. Heat until agar
is clear, do not boil. Reweigh flask with contents and add sterile water to adjust
total weight to compensate for any water loss during heating process. Place flask
in 50°C rotator until agar mixture cools to 50°C (approximately 2 hours). Add
28 ul ethidium bromide to agar mixture and mix well. Pour 25ml into previously
leveled agar tray and place 2 agar combs approximately 0.5 cm and 3.5 cm fi'om
left side origin. Makes approximately 14 gels.

. Once DNA specimen has been solubilized in TE Bufl’er at 56°C for a minimum
of 2 hours, vortex warmed specimen for 5 seconds before opening tube.

. Remove 4 ul of extracted DNA and combine with 2 ul loading solution.

. Combine 4 ul of Visual Marker (Lambda HINDIII/ECORI) with 2 ul loading
solution.

. DNA quantification standards must be used on each gel. These already contain
loading solution. Load 6 ul of each standard quantity (500ng, 250ng, 125ng,
63ng, 31ng, 15ng).

. Place gel into BRL mini-gel apparatus. Pour 250 ml TAE Bufi‘er into apparatus

and add 25 ul ethidium bromide to TAE Buffer.

58

10.

59

Load Lambda Visual Marker into top well, then DNA Quantification Standards
in order from highest quantity (500 L11) to lowest quantity (15 ul).

Load samples placing Lambda Visual Marker into top center well if a second set
of lanes are used.

Set the voltage at 175 volts and stop the run when the bromophenol blue tracking
dye in the load solution has moved 1 - 2 cm from the origin.

Remove the gel from the BRL mini-gel apparatus and photograph.

APPENDIX G

APPENDIX G

REVERSE DOT BLOT METHOD FOR GENETIC TYPING

. Prior to preparing the DNA test samples and PCR amplification, set GeneAmp®
instrument to appropriate PCR profile times and temperatures for 32 cycles of:
Denature: 60 seconds at 94°C, Anneal: 60 seconds at 60°C, Extend: 30 seconds
at 72°C, Final Step(Time Delay): 7 minutes at 72°C.

. Prepare DNA concentrations for amplification with TB Bufi‘er in the range of

2 to 10 ng in a total of 20 ul volume. Incubate samples at 56°C for 2 hours.

. Transfer PCR amplification reagents to a designated clean area. Place the required
number of tubes containing 40 ul of pre—aliquotted AmpliType® PM PCR
Reaction Mix into a rack. Label the reaction tubes.

. Ensuring the reaction solution is at the bottom of the tubes, pipet 40 ul of
AmpliType®PM Primer Set on the side of each tube, including control tubes.
Begin the cycling process within 20 minutes after the addition of the Primer Set.

. Add 2 drops of Mineral Oil to all tubes.

. Add 20 ul of extracted sample or control sample to corresponding tubes by
inserting the pipet below the Mineral Oil layer.

. Place the tubes into the GeneAmp PCR instrument, pushing the tubes completely
down into the sample wells. Start the 32 cycle amplification.

. After amplification, add 5 ul of 200 mM disodium EDTA to each tube.

60

61

9. Remove a 5 ul aliquot from each tube for post-amplification electrophoresis.

10. Prepare a 3% NuSieve®/ 1% SeaKem® agarose gel: Add 4 grams NuSieve

ll

12.

13.

14.

15.

16.

with 1 gram SeaKem agarose to 100 ml of 0.5X TBE Bufi’er in pre-weighed flask.
Heat agar until clear, reweigh flask and add sterile water to compensate for any
water loss during heating process. Add 5 ul of 10 mg/ml stock ethidium bromide
to the agar mixture. Cool the solution to 55 - 65°C. Pour the gel into a leveled
casting tray and add one comb to top of gel. Allow to cool to room temperature

prior to use (30 minutes).

. Add 250 ml of 0.5X TBE Buffer to the bufi"er tanks with 12.5 111 of ethidium

bromide to buffer.

Add 2 ul loading buffer to 5 ul of each sample to be analyzed and 2 ul loading
buffer to a 5 ul stock solution of 123 by Ladder (50 ng/ul).

Load the 123 by ladder plus load buffer into the first well of the amplification gel.
Pipet the samples to be analyzed, mixed with loading buffer into the remaining
wells.

Run the gel at 110 - 115 volts for approximately 2 hours until the bromophenol
blue front has migrated 4 cm down the gel to adequately separate the six bands of
interest on the gel.

Photograph the gel.

If six bands are present in the samples at the same six positions as the 123 bp

Ladder, proceed with DNA hybridization.

62

HYBRIDIZATION PROCEDURE:

1.

2.

10.

Preheat a water bath at 54 - 56°C at 50 to 70 rpm.
Warm Hybridization and Wash Solutions at 37 - 55°C. (All solids must be in

solution).

. Allow probe strips to warm to room temperature inside storage tube.

Prepare the GeneAmp® instrument to maintain a 95°C temperature.

Place the sample and control tubes in the GeneAmp® instrument when
instrument has achieved desired temperature.

Denature the sample and control tubes for 3 - 10 minutes.

Label all sample and control HLA DQAl and PM strips.

Add 3 ml of pre-warmed Hybridization Solution to each well of the
hybridization tray while tilting the tray toward the labeled ends of the strips.
Add 20 ul of denatured sample or control to respective wells. Place in rotating
water bath for 15 minutes.

Five minutes before the end of the hybridization step, prepare the Enzyme

Conjugate Solution.

Enzyme Conjugate Solution:

Number of Strips X 3.3 ml Hybe Solution = Volume of Hybe Solution

Number of Strips X 27 ul Enzyme ConjugatezHRP-SA = Volume of Enzyme

ConjugatezHRP-SA

6 3

11. Aspirate contents of each well, wipe tray lid and add 5 ml pre-warmed Wash
Solution. Rinse by gentle rocking and aspirate Wash Solution.

12. Add 3 ml of the Enzyme conjugate Solution into each well, cover with lid and
place trays into water bath for 5 minutes.

13. Aspirate contents of each well, wipe tray lid, and dispense 5 ml of pre-warmed
Wash Solution to each well. Rock tray gently, aspirate and add 5 ml Wash
Solution again. Rotate trays for 12 minutes in heated water bath.

14. Aspirate each well, wipe tray lid, dispense 5 ml pre-warmed Wash Solution into
each well. Rock gently, aspirate and add 5 ml Citrate Bufi’er to each well.

15. Cover trays and place on orbital shaker for 5 minutes. Prepare Color
Development Solution at this time.

Color Development Solution:
Number of Strips X 5 ml Citrate Buffer = Volume of Citrate Bufi’er
Number of Strips X 5 ul 3% Hydrogen Peroxide = Volume of Hydrogen Peroxide
Number of Strips X 0.25 ml ChromogenzTMB Solution = ChromogenzTMB

16. Remove trays fiom orbital Shaker, aspirate each well, add 5 ml Color
Development Solution.

17 . Place lids on trays and cover trays with aluminum foil to protect from light.

18. Rotate on orbital shaker for 10 minutes or more until “S” and “C” dot is visible.

19. Stop Color Development by rinsing each well with distilled water 2 - 3 times.

20. Photograph strips after recording results.

APPENDIX H

APPENDIX H

REAGENTS AND SUPPLIES

. REAGENTS FOR ETHANOL PRECIPITATION AND CENTRICON®
EXTRACTION METHODS

. Stain Extraction Bufl’er: Dissolve 1.21 grams TRIS base and 5.84 grams NaCl
into 500 ml (18 ohm) distilled water. Adjust pH to 8.0 with HCl. Add 100 ml
20 % SDS and 20 ml 0.5 M Na; EDTA-2H20. Bring to a final volume of 1.0
liters with distilled water. Store at room temperature.

Sterilize prior to use by filtration.

Note: Add 6.02 mg DTT/ml of stain extraction buffer prior to use.

. TRIS base: tris(hydroxymethyl)aminomethane, CJI11N03 , (Sigma® Trizrna
base S-8524 molecular biology grade).

SDS (20% w/v): Slowly dissolve 200 grams sodium dodecyl sulfate

(SDS), CH3 (CH2)110S03 Na, electrophoresis grade (ultra pure), Boehringer
Mannheim® 100155.

. Dithiothreitol (DTT 0.39M): Dissolve 601.2 milligrams DTT to 10 ml
distilled (18 ohm) water. Aliquot and store in fi'eezer.

. Proteinase K (10 mg/ml, 10 ml): In sterile, disposable plastic 15 ml tube
dissolve 100 mg Proteinase K (Sigma® P—49l4, molecular biology

grade) into 10 ml sterile, deionized water. Store 100 111 aliquots in

64

65

sterile tubes at -20° C. Thaw tubes as needed.
Discard unused portions of thawed tubes.
. Phenol/Chloroforrn/Isoamyl Alcohol (100/100/4): Melt phenol at 65°C
and pour 100 grams into a Bellco® glass bottle. Add 200 mg
8-hydroxyquinoline and mix the solution thoroughly. Add an equal
volume of 1 M TRIS, pH 7.5, transfer to a separatory funnel and mix.
After the phases have separated, drain the lower phenol layer into the
Bellco® bottle. Drain the upper aqueous layer into a waste bottle. Add
an equal volume of 0.01 M TRIS, pH 7.5 to the phenol, transfer to a
separatory funnel and mix. Capture the lower phase in a bottle. Capture
the upper phase and determine it’s pH. Ifthe upper phase is 7 .5, cease
equilibration. If the pH is < 7.5, repeat the extraction with 0.01M TRIS
until the pH is 7.5. Combine the equilibrated phenol with a solution of
100 ml chloroform + 4 ml isoamyl alcohol. Cover the solution with
0.1 M TRIS and store in the refiigerator.
. TE Buffer (Tris-EDTA Bufl’er): Dissolve 0.605 grams TRIS base (10mM) into
250 ml distilled water (18 ohm). Bring pH to 7.5 with HCl. Add 0.0185 grams
Na EDTA (0.1mM), adjust pH if required. Bring final volume to 500 ml with
500ml distilled (18 ohm) water. Autoclave.

Store at room temperature.

. Cold Absolute Ethanol (200 Proof) Quantum® Chemical.

66

B. REAGENTS FOR SLOT BLOT QUANTIFICATION
1. Spot Solution: Thoroughly mix 6 ml 5N NaOH, 3.75 ml 0.5 M EDTA, 150 111

0.04% Bromothymol Blue (provided in kit) and 65 ml of distilled (18 ohm)

water.
Stable for at least 3 months at room temperature.

2. Biodyne® B nylon membranes are supplied by BRL and used for
slot blot procedures using Chemiluminescence(ECL ®) probes for
Visualization. Store at room temperature.

3. Pre-Wet Solution: Mix 40 ml NaOH, 25 ml 0.5 M EDTA and
435 ml distilled (18 ohm) water.

4. Hybridization Solution: (5X SSPE - 0.5% w/v SDS, 1 liter)

Mix together: 250 ml 20X SSPE, 25 ml 20% w/v SDS and 725 ml
deionized water. Store at room temperature. Warming may be
necessary to dissolve solids in solution prior to use.

5. Hydrogen Peroxide (3 0% w/v).

6. QuantiBlot® D1721 probe (supplied in kit).

7. Wash Solution (QuantiBlot® 1.5X SSPE, 0.5% w/V SDS, 2 liters.

Thoroughly mix: 150 ml of 20 X SSPE, 50 ml 20% w/v SDS and
1800 ml distilled (18 ohm ) water. Warming may be necessary to

dissolve solids prior to use.

8. Enzyme Conjugate:HRP-SA supplied by Perkin Elmer ®.

67

9. ECL supplied by Amersham ® Life Science.
C. Yield Gel Reagents
1. Standards: (Store Lambda phage DNA at 250 ug/nrl in freezer).
Make serial dilutions with TE Buffer as follows by combining 1.0 ml
of diluted standards with loading solution as shown:
1.0 ml at 125.0 ug/ml + 0.5 ml loading solution = 500 ng/6u1
1.0 ml at 62.5 ug/ml + 0.5 ml loading solution = 250 ng/6ul
1.0 ml at 31.3 ug/ml + 0.5 ml loading solution = 125 ng/6ul
1.0 ml at 15.6 ug/ml + 0.5 ml loading solution = 63 ng/6ul
1.0 ml at 7.8 ug/ml + 0.5 ml loading solution = 31 ng/6ul
1.0 ml at 3.9 ug/ml + 0.5 ml loading solution = 15 ng/6ul
2.. Ethidium Bromide (5 mg/ml)
Dissolve 250 mg ethidium bromide (2,7-diamino-10-ethyl-9-
phenylphenanthridinium) into 45 ml deionized water. Adjust volume
to 50 ml. Store at 2 - 8°C and protect from light in brown bottle or
with aluminum foil.
Warning: Ethidium Bromide is a mutagen. Always wear gloves.
3. Sigma Type H agar supplied by Sigma® Chemical.
4. Lamda HIND III / ECOR l supplied by GIBCO® BRL.
5. Load Solution: (50% glycerol - 0.1% bromphenol blue- 0.1 M EDTA

in TE Buffer, 100 ml).

Thoroughly mix 50 ml glycerol, 20 ml 0.5 M EDTA, 30 ml TE Buffer

6 8

and 0.1 gram bromphenol blue. Store at 4°C.

6. TAE Buffer (20X)
Mix 96.6 grams TRIS base, 22.8 ml glacial acetic acid and 40.0 ml
0.5 M EDTA (pH 8.0). Bring to a final volume of 1.0 liter with water.
Store at room temperature.

D. Dot Blot Reagents

1. AmpliType® PM PCR Reaction Mix, Primer Set, Control DNA 1,
Mineral Oil, PM DNA Probe Strips, HLA DQAl Probe Strips,
Enzyme ConjugatezHRP - SA, Chromogen TMB supplied by
Perkin E1mer®.

2. Base Pair Ladder (123) supplied by GIBCO BRL®

3. NuSieve® GTC ® supplied by FMC, Rockland, ME.

4. 95% ethanol supplied by MLS.

5. DNA Typing Trays Perkin Elmer (Part no. N808-0065).

6. Aspirator apparatus.

7. Thermal Cycler 480 Perkin Elmer GeneAmp®.

8. Hybridization Solution (5X SSPE, 0.5% w/v SDS 1 liter).
Add 250 ml of20X SSPE to 25 ml of 20% w/v SDS into 725 ml
distilled (18 ohm) water and mix thoroughly. Warming may be
necessary prior to use.

9. Wash Solution ( 2.5X SSPE, 0.1% w/V SDS 2 liters).

Add 250 ml of20X SSPE and 10 ml 20% w/V SDS tO 1.740 ml

6 9
distilled (18 ohm) water and mix. Warming may be necessary.
10. Citrate Bufi’er
Dissolve 18 grams of trisodium citrate dihydrate (Na3C6H507 *2H20)
in 800 ml distilled (18 ohm) water. Adjust the pH to 5.0 by addition
of approximately 6 grams of citric acid, monohydrate (C5HsO7‘HzO).
Adjust to a final volume of 1 liter using distilled (18 ohm) water,

mix thoroughly and autoclave or sterilize by filtration.

APPENDIX I

APPENDIX I

GLOSSARY
ALLELE - One of several alternate forms of a gene at a given locus (location on the
DNA molecule).
AMINO ACIDS - The building blocks of which proteins are constructed.
AMPLIFICATION - Repetition of target sequence of DNA molecule by the enzyme
Taq polymerase.
ANNEAL - Binding of primer segment of DNA close to the area of interest.
BASES - Purines or pyrimidines in DNA that provide the genetic code which
determines the amino acid structure of proteins.
CHROMOSOME - A single DNA molecule which possesses genes, including
attached proteins that maintain structure of the DNA molecule.
DNA - Two intertwining molecular chains which consist of many nucleotide bases
linked together by sugar (deoxyribose) and phosphate groups.
DQAl - A location on chromosome 6 divided into four major types (DQAl, DQA2,
DQA3, DQA4) and three subtypes (DQA1.1, DQA1.2, DQA1.3).
EPIDERMIS - Surface layer of human skin containing stratified squamous epithelial
cells.
EXTENSION - Process in polymerase chain reaction afier annealing of primers which

. elongates the DNA chain with the addition of complementary bases.

70

71

GENE - The area on a chromosome consisting of many base pairs which is
responsible for physical and biochemical traits such as hair color or diabetes.
GENOME - All the genetic material within a cell or individual.

GENOTYPE - All or part of an individual’s genetic constitution.

HLA - Human leukocyte (white blood cell) antigens; human antigens that play a
major role in compatibility between tissues and some blood cells.

HLA DQAl - Also termed HLA DQa. A gene on chromosome 6 of the HLA system
consisting of 6 alleles resulting in 21 difi‘erent genotypes.

LOCI - Areas on a chromosome that occupied by a specific gene.

NUCLEOTIDE - Building blocks of nucleic acid. Each nucleotide consists of a
phosphate group, a sugar and either a purine or a pyrimidine base.
OLIGONUCLEOTIDE - A short section of a DNA strand used as a probe or primer
in the polymerase chain reaction.

POLYMORPHISM - Differences in arrangement in amino acid sequence which form
the basis of self recognition.

RESTRICTION ENZYMES - Bacterial by-product which makes sequence specific
cuts along the DNA chain according to the sequence the enzyme identifies.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM - Production of DNA
fiagments of difi’erent lengths by a restriction enzyme due to inherited differences

along the DNA molecule.

LIST OF REFERENCES

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Blake, E., Mihalovich, J ., Giguchi, R, Walsh, P.S., & Erlich, H. (1992). Polymerase
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