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DNA TYPING OF TEN POSTMORTEM PSOAS MUSCLE SAMPLES
USING THE RESTRICTION FRAGMENT LENGTH POLYMORPHISM
TECHNIQUE AND CHEMILUMINESCENCE

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Amélia C. Harlukowicz

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DEOXYRIBOSE NUCLEIC ACID (DNA) TYPING OF TEN POSTMORTEM
PSOAS MUSCLE SAMPLES USING THE
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
TECHNIQUE AND CHEMILUMINESCENCE

By

Amelia Christy Harlukowicz

A THESIS

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

MASTER OF SCIENCE

School of Criminal Justice

1996

ABSTRACT
DNA TYPING OF TEN POSTMORTEM
PSOAS MUSCLE SAMPLES USING THE
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
TECHNIQUE AND CHEMILUMINESCENCE
by

Amelia Christy Harlukowicz

The task of identifying human remains at a crime scene, can be
difficult due to the decomposition of biological tissues. Traditional means of
identification, such as fingerprints, x-rays and dental records are useless if a
body is badly decomposed. DNA evidence provides a strong statistical link
between biological evidence and an individual from a crime scene.
Unfortunately, the lack of sample and/or degradation of DNA produces poor
or even no results using restriction fiagment length polymorphism (RFLP)
analysis. Several studies have evaluated the DNA recovered from various
biological tissues. High molecular weight DNA has been successfully
recovered from psoas muscle. The psoas muscle aids in flexing the thigh
laterally. Muscle fibers carry out limited enzymatic reactions. Enzyme
activity tends to shear DNA into small fragments and even shorten the DNA
fi‘agment lengths. Ten psoas muscle samples were evaluated using the
Lifecodes DNA typing protocol. Chemiluminescent labeled probes reduce
safety concerns and speed up result time. Nine of the ten samples tested

provided useful DNA typing results within a 16 hour time period.

Copyright by
Amelia Christy Harlukowicz
1996

To Elaine, Carrie, and Ann Marie Harlukowicz
“It’s a great life if you don’t weaken”

ACKNOWLEDGMENTS

This thesis would never have been completed without the manpower
and persistence of Dennis Keagle. His time and knowledge were an integral
part of this research. I would also like to thank Dr. Joseph Melvin and
Sparrow Hospital. Without their resources this project would not have been
possible.

My sincere appreciation also goes to Bill Dean and Dorothy Dean.
They came to my rescue when an end to this research project seemed
unattainable. Dr. Roger Smith, Raj Patel and the University of Cincinnati
Hospital were also significant resource providers in this research.

I would like to thank Joan Burke. Her patience and understanding
while I completed this thesis have been very much appreciated. Her
numerous attempts at proofreading my work were very invaluable.

Thanks are also extended to my family and friends. You all stuck with
me even when I did not believe I could stick this out myself.

Finally I would like to thank Dr. Jay Siegel for his guidance and
support while I was learning the thesis process. His guidance during my

graduate career and job hunting quest was very valuable.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. vii
LIST OF FIGURES .............................................................................. viii
CHAPTER 1
INTRODUCTION ....................................................................................... 1
CHAPTER 2
LITERATURE REVIEW ............................................................................. 12
CHAPTER 3
METHODOLOGY ....................................................................................... 20
CHAPTER 4
FINDINGS .................................................................................................. 29
CHAPTER 5
CONCLUSIONS & RECOMMENDATIONS .......................................... 36
APPENDICES
Appendix A DNA isolation from muscle tissue ................................ 39
Appendix B Determination of quantity and quality from
yield gel .................................................................................. 41
Appendix C Restriction digestion of isolated DNA ......................... 43
Appendix D Determination of completeness of digestion
from test gel ........................................................................... 45
Appendix E Analytical gel electrophoresis ...................................... 48
Appendix F Procedure for preparation of agarose gels ................... 50
Appendix G Preparation of transfer working solutions .................. 52
Appendix H Southern transfer of DNA onto nylon membrane ...... 53
Appendix I Hybridization of nylon membranes .............................. 55
Appendix J Reagents ....................................................................... 58
Appendix K Computer assisted lumigraph analysis ...................... 61
Appendix L Function of select reagents and controls .................... 66

LIST OF REFERENCES ........................................................................... 69

LIST OF TABLES

Table 1-Age of psoas muscle samples .............................................. 25
Table 2-Isolation and allelic control ranges .................................... 28
Table 3-Results of computer assisted lumigraph analysis ............. 30
Table 4-Results of yield gel interpretation ...................................... 32
Table 5-Results of test gel interpretation ....................................... 34
Table 6-Preparation for restriction digestion ................................. 42

LIST OF FIGURES

Figure 1-Lumigraph developed with LumiPhos 480 ....................... 31
Figure 2-Yield gel photo .................................................................... 33
Figure 3-Test gel photo ...................................................................... 35

INTRODUCTION

Often times the only evidence available at a crime or death scene is the
presence of human remains. The identification of human remains using DNA
testing offers many advantages and few disadvantages to law enforcement
agencies and the legal system. Traditional genetic marker testing is not very
successful in associating or disassociating suspects with a crime. DNA
evidence provides a strong statistical link between biological evidence and an
individual from a crime scene.

The primary function of forensic biology is to establish associations
between biological samples and the individuals from which they originated
(W aye et. al., 1993). Advances in deoxyribonucleic acid (DNA) technology
over the past decade have given scientists the ability to better identify such
individuals. DNA evidence has a very high probative value. Except for
identical twins, the DNA of each individual is unique. More than 99% of the
DNA genetic code is the same for all people. This 99% is what makes us
human beings. Forensic scientists are interested in the remaining one

percent which is responsible for the variations among individuals.

2
Forensic analysis of DNA is an extension of classical serology methods

that have been performed for more than 50 years. Karl Landsteiner, in 1902,
first suggested that ABO typing could be used to associate or exclude blood at
a crime scene to a suspect. The ABO system was first used to determine
family relationships. Later this technique was applied forensically.
Conventional typing systems only provide limited statistical information to
individualize crime scene samples. The ABO blood group system, for
example, only discriminates between four types (A, AB, B and 0). DNA
typing provides the potential for greater statistical individualization. Some
have claimed that the use of DNA profiling has been the biggest
breakthrough in the forensic science community since fingerprinting was
developed.

Although this paper focuses on forensic DNA analysis for identification
purposes, this procedure has been successful in other forensic disciplines
including medicine, paternity testing, animal and plant science and wildlife
forensic science (Kirby, 1992). Forensic DNA typing is simply the application
of DNA typing techniques from the medical research arena to matters
concerning criminal justice and the forensic science community.

Forensic DNA analysis can assist in solving serial crimes, determine if
more than one person was involved in a crime, place a suspect at a crime

scene even if there are no eye witnesses, eliminate falsely accused individuals

3
and even confirm persons suspected in insurance fraud cases (Lifecodes,

1990).

The task of identifying human remains may prove to be difficult due to
the decomposition of biological tissues. Traditional means of identification,
such as fingerprints, x-rays and dental records are useless if a body is badly
decomposed. In this instance, visual confirmation of a victim’s identity is not
possible.

A critical point in the development of useful DNA typing results is the
condition of a sample’s DNA. Contamination and degradation are two factors
to consider. A problem discussed in the literature is that lack of sample
and/or degradation of DNA produces poor or even no results using RFLP
analysis. RFLP analysis requires a sufficient quantity (>50 ng) of relatively
undegraded DNA. The FBI prefers at least 100 ng of relatively undegraded
DNA for analysis (Adams, 1988).

Degradation can result from several factors including humidity,
sunlight, bacteria and soil. The quality of results is dependent upon the
degree of degradation. The more severe the degradation the lower the
molecular weight of the DNA. Severely degraded DNA will produce no
results. A valid DNA pattern can be identified if partially degraded DNA is
of high enough quality to test. Inconclusive results are often the product of

typing partially degraded DNA.

4
The purpose of this research is to determine the quality and quantity

of DNA recovered fiom multiple postmortem psoas muscle samples. This will
be accomplished using the RFLP technique and chemiluminescent
development methods.

Due to the fact that high molecular weight DNA is a prerequisite to
obtain successful DNA typing results, it is important to investigate the
postmortem stability of DNA. Several articles have dealt with investigating
the results of DNA analyzed fi'om various biological tissues. A recurring
problem in the literature (discussed in the next chapter) is the fact that not
all tissues produce high-grade DNA results. Several studies have been
performed to determine the stability of DNA in various human organ tissues.
Muscle tissue, particularly psoas muscle, has proven successful in previous
studies.

The psoas muscle aids in flexing and rotating the thigh laterally. The
word psoa means muscle of loin (V anDeGraff, pg. 350). Skeletal muscle
tissue is responsible for various body movements. This group of muscle
contracts to carry out various body motions, produce heat, and provide good
posture. Muscle fibers carry out very little enzymatic activity. Other
biological tissue samples such as liver, carry out numerous enzymatic
reactions. Enzyme activity tends to shear DNA into small fi'agments and

even shorten the lengths of DNA fragments (Bar et. al., 1988). Therefore,

5
due to the fact that muscle tissue carries out only a limited number of

enzymatic reactions, it should provide good DNA typing results.

DNA contains our genetic blueprint and is found in every nucleated
cell in the body. The human genome is composed of 46 chromosomes.
Twenty-three chromosomes are inherited fiom the father’s sperm and 23 are
inherited from the mother's egg. DNA has several features that make it
useful for forensic purposes. One feature previously mentioned is the fact
that every individual has unique DNA except identical twins. One's DNA is
constant throughout their lifetime. In addition, DNA contained within an
individual's blood, semen, saliva, bone and every other nucleated cell is the
same.

Along with the numerous advantages of DNA evidence there are some
limitations. For example, statistical data relayed to a jury can often be
overwhelming and can overshadow other evidence. DNA evidence is not
individual. Statistical evidence may show that only a limited number of
individuals in the population share the same DNA pattern. However, DNA
analysis cannot be used to individualize evidence left at a crime scene to
exclusively one individual. DNA testing is very costly when analysis, legal
fees and expert witness expenses are all considered. This may prove to be a
disadvantage to some defendants. Finally, crimes are not solved solely fiom

DNA evidence. Even though DNA evidence has become increasingly popular,

6
some types of physical evidence such as fingerprints, may be more probative

(Genetic Witness, pg. 101).

There are two types of DNA analysis; restriction fi'agment length
polymorphism (RFLP) and the polymerase chain reaction (PCR). The RFLP
technique requires relatively high molecular weight DNA to produce useful
results. When a sample is of insufficient quantity or is degraded, PCR would
prove useful.

The PCR method is actually the amplification of small amounts of
DNA to produce a large quantity of useful DNA. The DQ-alpha typing
method, in conjunction with PCR, provides limited discrimination between
samples. The RFLP method requires a much larger sample than the PCR
method, but is a better tool for individualization. Thus choosing between the
two methods depends on whether sensitivity or specificity is the ultimate
goal.

Forensic DNA typing currently relies on variable regions of DNA called
variable number tandem repeats (VNTRs) or minisattelites. Tandem repeats
are small sections of repeated DNA sequences. These regions are repeated a
variable number of times. VNTRs vary greatly in size amongst the
population. A core sequence of bases can be present as a single copy and
repeated anywhere from 2 to 100 times. Jeffreys and his colleagues in 1985
used a sequence found in the myoglobin gene to describe properties of VNTR

sequences. Highly variable or polymorphic regions of DNA are analyzed

7
using the RFLP procedure. A DNA “fingerprint” obtained from this

procedure can prove to be a powerful investigative tool.
History

A Swedish biologist, Johann (Friedrich) Miescher, discovered an
unusual phosphorous containing substance in 1868. Miescher was studying
the nuclei isolated from the white blood cells from wounded soldiers
bandages. He named this material “nuclein.” “Nuclein” contained both
acidic and basic components. The acidic portion was DNA and the basic
portion was made of histones. Miescher found a similar “nuclein” substance
in a study he performed on salmon spermatozoa. This soon became the first
realization the DNA was a universal substance (Kobilinsky, 1993).

DNA is a nucleic acid made up of subunits known as nucleotides. The
chemical make up of DNA is phosphoric acid, deoxyribose (sugar) and four
nitrogenous bases. The adenine (A) and guanine (G) bases are purines,
cytosine (C) and thymine (T) are the pyrimidine bases.

Erwin Chargofl‘ discovered in 1947 that the purine to pyrimidine ratio
(A+G)/(C+T) in DNA was always equal to 1.0. Each pair of bases is bound by
a hydrogen bond and is referred to as a base pair (bp). In 1950 Chargoff and
his colleagues discovered a critical point in determining the DNA structure.
Their findings were that for every one mole of adenine there is one mole of
thymine and for every one mole of cytosine there is one mole of guanine

(W aye et. al., 1993).

8
In 1953, James Watson and Francis H. C. Crick presented a paper in

Nature revealing the structure of the DNA molecule. Their model depicted
the DNA molecule as a right-handed double helix. The double helix is formed
by a phosphate sugar backbone. Along this backbone the four different
nitrogenous bases are attached. This model somewhat resembles a ladder
and its rungs. The bases can form any number of sequences within a single
strand of DNA. The two chains of the DNA double helix run in opposite
directions. If one chain runs from the 5’ to the 3’ end then the
complementary chain containing the complementary base sequence will run
3’ to 5’. For example, if one DNA strand was 5’-A-C-T-A-G-T-C-3’ then the
complementary chain would be 3’-T-G-A-T-C-A-G-5’. The 5’ and 3’ numbers
indicate the numbering across the carbon atoms of the sugar molecules.
Many of the techniques used to analyze DNA are based on the
complementary base-pairing of the DNA molecule.

DNA analysis has a history that dates back before its use to identify
crime scene samples. David Botstein and his colleagues were the first to use
the RFLP procedure in 1980 to begin to construct a human gene map. There
is no difference in the procedures used for clinical DNA analysis and forensic
application.

The RFLP procedure was first applied forensically by Jeffreys in 1985.
Jeffrey’s procedure used a variable number tandem repeat (VNTR) probe

That detected multiple VNTR loci simultaneously. This multilocus technique

9
produces results that are very complex. Although this is a good

discriminating technique, it is oftentimes diflicult to analyze mixed samples.
Mixed samples contain DNA from more than one individual and are often
encountered in forensic cases. Another potential problem is the fact that
multilocus analysis is not a good technique to use on degraded DNA or
limited amounts of sample (Waye et. al., 1993). A vast majority of
laboratories in the USA and Europe use single locus probes. Single locus
probes have proven to be more sensitive and their DNA profiles are easier to
interpret (Balazs, 1992).

The United Kingdom, in 1985, was the first country to use DNA typing
for forensic purposes. In the United States, DNA testing was introduced to
private and state laboratories in late 1986. The Federal Bureau of
Investigation (FBI) began DNA casework in 1988. Today, virtually all state
and federal courts accept DNA evidence. The few instances in which DNA
was inadmissible were due to disputes over statistical matters and not due to
the validity of the DNA testing techniques themselves. The majority of the
arguments concerning DNA testing laboratories are how certain testing is
performed (Ohio, 1994).

Laboratory standards and guidelines have been developed by the
Technical Working Group on DNA Analysis Methods (TWGDAM). TWGDAM
meets several times a year and is made up of scientists fi'om the industry,

forensic science and academic fields. TWGDAM has published guidelines to

10
assure quality assurance among DNA laboratories. The admissibility of DNA

test results into court often depend upon whether TWGDAM guidelines were
followed.

The National Research Council (NRC) issued a report in 1992 detailing
how various parties in the legal system could be affected by DNA evidence.
Their major conclusions are as follows. The main purpose of any scientific
analysis is to provide as much information as possible on which guilt or
innocence can be determined by a jury. As with any expert testimony, the
DNA expert must keep matters as simple as possible when addressing a jury.
It is important that the jury does not become overwhelmed with statistics,
scientific terms and techniques. Therefore, it is suggested that the DNA
experts use charts and reports to thoroughly educate the jury about DNA
typing evidence.

Prosecutors have the responsibility of making defense counsel and
experts aware of all evidence necessary to perform DNA tests. The NRC also
suggests that any materials such as the experts' data sheets and other
materials be readily available for use in court to ensure thorough evaluation
of all scientific evidence.

The prosecutor also has the responsibility of informing defense
counsel early on that DNA testing is involved. This allows defense counsel

to acquire any necessary expert assistance. A portion of the DNA sample

11
should be preserved for repeat testing whenever possible. This allows the

defense to perform its own analysis of the evidence.

LITERATURE REVIEW

In 1985, Jefi‘reys, Wilson and Thein discovered that the human
genome contains polymorphic “minisatellite” regions. These regions have a
10-15 base pair (bp) ‘core’ sequence. Probes were designed that detected
multiple “minisatellite” regions simultaneously. Their research would later
prove to be the beginning to DNA “fingerprinting” as it is known today.

As a follow-up to their previous study, Jefl'reys, Wilson and Thein
(1985) looked at the DNA fingerprints of twenty unrelated British
Caucasians. They were successful in their attempt to illustrate the DNA
patterns were highly specific to each individual.

Then in October 1985, Gill, Jeffreys and Werrett published a study
that showed that DNA fingerprinting could be used for forensic purposes.
This was the first major article to deal with the application of this technique
to biological samples in forensic science. An attempt was made to show that
DNA fingerprinting could prevail over conventional serologic methods used to
analyze various biological samples such as blood and semen.

In 1986, Kanter, Baird, Shaler and Balazs analyzed RFLPs from DNA
recovered from dried bloodstains. This study showed that high molecular

weight (HMW) DNA could be recovered from dried bloodstains as old as three

12

13
years. Blood stains that enter a laboratory may be dried, aged or

contaminated to the point where results obtained from traditional genetic
typing may be unsuccessful.

A similar study was performed by Guisti, Baird, Pasquali, Balazs and
Glassberg (1986). This study focused on DNA recovered from sperm. The
authors presented evidence that DNA could successfully be extracted fiom
spermatozoa to perform RFLP analysis. The RFLP patterns of both a
subject’s sperm and blood DNA were identical. The results of this study
demonstrated the reproducibility of RFLP analysis and the possibility of
excluding or including assailants in rape cases.

Gill, Lygo, Fowler and Werrett (1987) evaluated DNA fingerprinting
for forensic purposes. The results of this study are fourfold: 1) HMW DNA
can be isolated from blood and semen stains up to four years old. 2) The
positive identification of rapists is possible. Sperm nuclei and vaginal debris
are first separated from mixed stains. The two different fractions can then be
analyzed separately. 3) Vaginal DNA can be isolated fi'om penile swabs and
then analyzed. 4) DNA can successfully be obtained fi'om buccal swabs.

In 1987, Baird, Giusti, Meade, Glyne, Shaler, Bemm, Glassberg, and
Balazs of Lifecodes Corporation applied DNA typing to forensic biological
materials. RFLP analysis was performed on 170 forensic samples. Samples
included kidney, liver, muscle, brain, blood and semen. They also reported

the findings of some forensic cases. One case in particular involved a

14
homicide victim. An abandoned car was found with what appeared to be

brain fragments on the front grill. The car’s owner was missing and no body
was recovered. The DNA recovered from the victim’s parents’ blood was
consistent with having the same pattern as the victim’s DNA. Statistical
analysis showed that the DNA isolated from the brain tissue was 160,000
times more likely to have come fi'om the ofi'spring of the two parents than
from any other individual.

Swarner, Reynolds and Sensabaugh (1989) evaluated the quantity of
DNA from postmortem heart, spleen, liver, muscle, blood, hair and bone. The
purpose of their research was to determine which postmortem tissue
produced the best quantity and quality of DNA. The goal of this study was to
determine if one type of tissue would be the best source of HMW DNA.
However, all the tissues yielded DNA. No one tissue proved to be a
reproducible source of HMW DNA. Another finding of this research was that
no correlation could be drawn between the time of death and the quality of
DNA results from each sample. They suggested that a collection of several
tissues would be useful to ensure successful DNA typing results.

Bar, Kratzer, Machler and Schmid (1988) published an article on the
postmortem stability of DNA. The major focus of DNA typing in this study
was identification purposes. They concluded that, “the amount of degraded
DNA correlated directly to the duration of the postmortem period.” (Bar et.

al. page 59). Due to the fact that relatively HMW DNA is required to obtain

15
reliable RFLP results they studied postmortem stability of DNA under

various conditions. Changes of banding patterns at various postmortem
intervals were studied using tissue specimens of brain, lymph nodes, liver,
spleen, psoas muscle, kidney, thyroid gland and blood. They stated, “the
discovery of DNA polymorphism’s represents a novel and powerful means of
identification of bodies by reconstructing familial relations.” (Bar et. al. page
68). Relatively HMW DNA was recovered in high quantities from blood,
psoas muscle, brain cortex and lymph nodes. Good DNA stability up to 5
days postmortem was observed in kidney, spleen and thyroid gland tissues.

In 1988, Higuchi, Von Beroldenger, Sensabaugh and Erlich typed the
DNA from single hairs. Their approach was different from the previously
discussed RFLP method of DNA typing. RFLP analysis requires a relatively
large amount of undegraded DNA for typing. Often times such DNA cannot
be obtained fiom such forensic evidence as blood stains and single hairs.
This study utilized the polymerase chain reaction (PCR) to amplify the DNA
from single hair roots. The findings were that any DNA from degraded
biological samples, that are not suitable for RFLP analysis, can be analyzed
using PCR. These samples would include old blood or semen stains and
possible mummified tissue remains.

An important study was completed in 1989 by McNally, Shaler, Baird,
Balazs, DeForest, and Kobilinsky. They analyzed the effects of various

environmental insults on the RFLP patterns obtained from DNA. This study

16
evaluated results of DNA fi'om bloodstains exposed to ultraviolet light, heat,

humidity, and soil contamination. The quality of the DNA did not result in
false RFLP patterns under the conditions studied. One significant effect
observed however, was the weakening of the overall RFLP pattern produced.

In 1989, a study was performed by Honma, Yoshii, Ishiyana, Mitani,
Kominami, and Maramatsu. This research used the DNA fingerprint
technique to characterize semen samples. By comparing DNA results from
blood and semen of several volunteers, the authors successfully determined
the semen donors. The semen from a condom left at a crime scene was also
analyzed in this study. A person was found dead in a Tokyo hotel room.
Beside her body lay a condom. The DNA from the semen in the condom and
the blood of a person suspected of the murder were the same. This was the
first time the DNA typing technique was successfully used in Japan for
investigative purposes.

Yokoi and Sagisaka (1990) presented an article on the postmortem
stability of DNA retrieved fi'om human bloodstains and various other
biological tissues. DNA was obtained from liver, lung, heart, pancreas,
spleen, kidney, adrenal gland, thyroid gland, psoas muscle, brain cortex and
lymph node tissues. High molecular weight DNA was retrieved fi-om the
heart, thyroid gland, psoas muscle and brain cortex. The high molecular

weight DNA fiom the liver, pancreas, spleen, lung, and kidney degraded

17
quite rapidly. The retrieval of high molecular weight DNA fiom bloodstains

was successful for stains up to two years old.

A case report was published in 1990 on the identification of
decomposed human remains using DNA profiling. Haglund, Reary, and
Tipper were the first to report the use of DNA fingerprinting using DNA from
the victim’s parents to establish identity. The victim was in a moderate to
advanced state of decomposition. Visual, x-ray and fingerprint confirmation
were not possible. Successful DNA results were obtained from three known
blood samples and the victim’s psoas muscle. This case demonstrated that a
“valid” option for identification was that of comparing DNA fiom the
deceased to relatives.

In 1991, Lee, Pagliaro, Berha, Folk, Anderson, Ruano, Keeth, Phipps,
Herren, Gainer and Gaensslen used DNA analysis to study genetic markers
in human bone. The quantity and degree of DNA degradation were studied
using spectrophotofluorometry and ethidium bromide. They found that the
quantity of DNA recovered is dependent on bone type. It was estimated that
the amount of DNA recovered from spongy tissue was 10 to 20 times greater
than that recovered fi'om compact bone. The ethidium-bromide method
proved more useful when estimating the HMW DNA content than the
flourometry method. This study established that bone tissue is suitable for

both for RFLP and PCR analysis. The results from this research also

18
confirmed that DNA patterns from blood and bone of one individual were the

same.

Schwartz, Schwartz, Mieszerski, McNally and Koblinsky (1991)
studied the quality and quantity of DNA obtained from dental pulp of human
teeth. The teeth were exposed to various environmental factors including
soil, seawater, humidity, variation in pH, temperature and burial. RFLP
analysis was performed on the samples and it was found that soil was the
only environmental factor that affected the DNA results. DNA was also
successfully isolated fi'om a 19-year old tooth in this study.

Also, in 1991, Hochmeister, Budowle, Borer, Eggmann, Comey and
Dirnhofer analyzed DNA extracted from compact bone tissue from badly
decomposed bodies from known and unknown human remains. Successful
RFLP results were obtained in some cases, but the DNA was often too
degraded to produce successful RFLP patterns. PCR was successfully used
on some bone tissue up to 11 years old. The DNA recovered from the
decomposed remains was not correlated with the time of death. The DNA, in
some samples was so degraded that it was not suitable for RFLP or PCR
typing. This study provided useful data to support the potential reliability of
PCR typing for identification purposes when RFLP analysis proved
unsuccessful.

In 1993, Sprecher, Liss and Schumm published an article on Southern

transfer and chemiluminescent detection. The authors discussed the

19
simplicity and speed of chemiluminescent detection over radioactive detection

methods. Using a nonradioactive detection method they detected a 10 ng

sample of human genomic DNA within seven hours.

METHODOLOGY

The experiments were conducted in several stages. Equal emphasis
was placed on both qualitative and quantitative results. The goal in this
research was to assess the reproducibility of the RFLP results from a number
of different samples. The method was to use the RFLP typing technique on
ten different psoas muscle specimens.

The RFLP procedure involves the following steps developed by Edwin
Southern in 1975: 1) isolation of genomic DNA from biological samples, 2)
determining the quantity and quality of DNA, 3) digesting the DNA with a
suitable restriction enzyme, 4) separating the DNA fragments using gel
electrophoresis, 5) transferring the DNA fragments from an agarose gel to a
nylon membrane using Southern transfer, 6) exposing the membrane-bound
DNA fragments to labeled probes, and finally, 7) developing the membrane
with x-ray film (NRC pg. 38).

Genomic DNA can be isolated fi'om many different substrates. The
isolation process usually involves placing the cellular material in a buffered
solution of salts, detergents and proteinase K. The cells are lysed by the

detergents and the cellular proteins are digested by the proteinase K. Any

20

21
extraneous debris is removed and genomic DNA is precipitated out with

ethanol.

The quantity and quality of genomic DNA is determined once isolated
from the forensic specimen. An agarose “yield gel” is used in this step.
Unknown DNA samples and control samples are loaded onto an agarose gel.
The gel is stained with ethidium bromide. After electrophoresis, ultraviolet
light is used to visualize the DNA. High molecular weight (HMW) DNA
suitable for RFLP typing will exhibit a HMW band greater than 20 kilobases
(kb). If the DNA has been degraded, a smear of DNA forms. The extent of
the smear is proportional to the degree of degradation (Saferstein, 1993).

Restriction enzymes cleave the DNA at specific sequences. These
enzymes are sometimes referred to as chemical scissors. There are numerous
restriction enzymes but the two used most fi'equently in forensic DNA typing
are HaeIII that recognizes the sequence 5’-GGCC-3’ and PST I that
recognizes the sequence CTGCAG. Restriction enzymes are also chosen
based on what probes are compatible with that enzyme. The process of
cleaving the DNA molecule is called digestion. An agarose “ Re test gel”
stained with ethidium bromide can determine the completeness of digestion.

Once the DNA has been digested, the fragments are separated
according to size by electrophoresis in which an electrical current runs
through the agarose or acrylamide gel forcing the smaller fragments to move

faster through the gel while the larger fragments remain behind.

22
Agarose and acrylamide gels are very fragile and they can easily be

damaged during the hybridization, wash and autoradiograph steps. A
Southern transfer must be performed before handling the gels, to transfer the
DNA fragments to a nylon membrane. This principle uses capillary action
to transfer the agarose bound DNA to the nylon membrane. Southern
transfer or Southern blotting, as it is sometimes referred to, was first
described by Edwin Southern in 1975 (Kirby 1992). Once the Southern
transfer is complete, the fragments are denatured. Denatured DNA is single
stranded.

The hybridization step involves recognition of a particular DNA
sequence with a complimentary sequence called a probe. Probes are single
strands of DNA that contain a specific base sequence. For example, a probe
with the sequence “ACTGA” will bind to the complementary sequence
“TGACT”. Probes can be either radioactively (32P) labeled or
nonradioactively labeled. Hybridization conditions need to be correct to
permit the probe to bind to the membrane bound DNA fi'agments. A washing
process follows the hybridization process. This allows excess unbound probe
to be washed away. Once the membranes are washed they are placed next to
a sheet of x-ray film in a cassette. Autoradiographs of membranes hybridized
with 32F can take one to five days to develop (Farley pg. 44). An advantage of

using non-radioactively labeled probes is speed of development. Kirby states

23
that within one hour a signal fi'om one ng of DNA can be detected with

chemiluminescent labeled probes (Kirby, 1992).

The research was completed following the Lifecodes RFLP
chemiluminescent procedure. Two single locus probes were chosen from the
Lifecodes protocol. Only two were chosen because of research costs and, in
addition, the two probes selected provided enough data to be meaningful.
The particular probes chosen for this research detect small and large
fi'agments of DNA.

As discussed earlier, probes are tagged pieces of DNA. They are
genetically-engineered to bind to single stands of DNA that contain
complementary base pair sequences. DNA probes are given specific names to
identify the DNA region, locus or gene to which the probe binds. For
example, probe D18827 can be dissected as follows: D18 signifies the DNA
sequence located on chromosome #18, the S indicates a single occurrence on
the chromosome and the 27 signifies sequence #27 registered in the Human
Gene Mapping database.

DNA probes can be polymorphic or monomorphic. Polymorphic probes
detect regions in the DNA molecule that vary dramatically in every
individual. Monomorphic probes detect DNA regions that are identical from
person to person. Polymorphic probes are used to establish DNA profiles for

identification purposes. The following is a list of some, but not all, of the

24
polymorphic probes used at Lifecodes: D2844, D17S79, D14813, D18SZ7,

DXYSl4, and D4Sl63.

For this research, two polymorphic probes were chosen based on the
size of the DNA fiagment each detected. The two probes selected were,
DIZSll and D17S79. Identity probe D12Sll has an allelic size range of
30-260 kilobases (Kb) long. Identity probe D17S79 has an allelic size range
of 2.0-7.0 Kb.

DNA isolation from muscle tissue

DNA was isolated from the postmortem muscle tissue samples using
the Lifecodes protocol in Appendix A. The approximate age of the psoas
muscle samples can be found in Table 1. The muscle tissue samples and
reagents used during the isolation process were kept on ice during use.
Determination of DNA quantity and quality from yield gel

The concentration of DNA in each sample must be known for a
successful restriction digest. Appendix B contains the steps performed
during this step of the RFLP procedure. A yield gel was prepared by
following the instructions in Appendix F, “Procedure for Preparation of
Agarose Gels.” Yield gel concentration results are found in Figure 1.
Restriction digestion of isolated DNA

Appendix C is the Lifecodes protocol that was followed during the

restriction digestion step of the RFLP procedure.

25

Table 1-Age of psoas muscle samples

 

Sample number

Hours before specimen was collected

 

 

 

 

 

 

 

 

 

 

 

1 Within 24 hours between 6/25/95 and 6/26/95
2 Within 24 hours between 6/19/95 and 6/20/95
3 Approximately 24 hours
4 Approximately 24 hours
5 Approximately 24 hours
6 Approximately 24 hours
7 Approximately 48 hours
8 Approximately 24 hours
9 Approximately 30 hours
10 Approximately 30 hours

 

 

Determination of the completeness of digestion from test gel

A test gel is run to determine if the samples have enough DNA to be
loaded onto the analytical gels in the next step of the procedure. Appendix F
is the protocol for preparation of a test gel. Appendix D contains the detailed

instructions that were followed during this stage of the procedure.

Analytical gel electrophoresis

The instructions for the preparation and assembly of analytical gels
are contained in Appendix F. The analytical gel is electrophoresed during

this step to separate the DNA fragments. Appendix E lists the detailed steps

followed during this process.

 

26
Southern transfer of DNA onto nylon membrane

Appendix G contains the preparation instructions for transfer working
solutions. After the electrophoresis of the analytical gels was complete, a
Southern transfer was completed to transfer the DNA from the agarose gels
onto nylon membranes. Appendix H contains detailed steps that were
performed during this procedure.

Hybridization

After a Southern transfer was complete, the membranes were
hybridized with the D12Sll and D17 S79 probes. Appendix I lists the
detailed steps followed during this procedure. After the hybridization
procedure was complete, the nylon membranes were placed in development
folders and sprayed with Lumi-Phos 480. The folders were placed into X-ray
cassettes and exposed overnight at room temperature. The X-ray films were
developed and the Lumigraphs were visually inspected.

Visual inspection of Lumigraphs

The Lumigraphs must be visually inspected to determine if the
following four criteria are met. 1. Were the samples completely digested?
There must be evidence of partialling. Extra alleles on the gel may be
present if the DNA was partially cleaved by the endonuclease. Extra bands
can also be present as a result of star activity. Star activity occurs as a result
of extra activity of the endonuclease. 2. The Lumigraphs must be observed
to determine if band shifting occurred because of salt concentration,
proteinase K, improper dilutions, or contamination. 3. Only one or two or
bands should be present in each lane. 4. Were the Lumigraphs exposed

properly? The membranes may have to be developed several times to

27
distinguish between close bands or to visualize all the alleles.

Computer assisted Lumigraph analysis

Even though a visual inspection may be sufficient to determine the
allele pattern of each sample, a computer is used to digitize the DNA bands
and compare them to controls. The internal controls are of known size for
DNA fragment size determination. The digitizing process has certain criteria
that have to be met to ensure the accuracy of the operator. An operator reads
the bands in a series of standards and controls with known band sizes. The
standards must be within 2% of the known DNA fragment sized. The
operator must re-read the standards if this criterion is not met. If the
controls do not fit within an acceptable range, the Lumigraph is considered
invalid and must be rerun. The control ranges for the Isolation control and
Allelic control can be found in Table 5. The results of the computer assisted
analysis for the psoas muscle samples can be found in Table 6. The
computer assisted analysis is actually designed to visualize bands and
perform calculations for paternity cases. Appendix J fully explains the

theories behind the calculations used in the computer analysis.

28

Table 2-Isolation and allelic control ranges

 

Control Ranges

Isolation Control

Allelic Control

 

 

 

 

 

D12S11 12.75-12.25 16.08-15.45
7.30-7.01 7.67-7.37

D17S79 3.92-3.77 4.12-3.96
3.50-3.37 3.63-3.49

 

 

 

 

FINDINGS

This research produced four major findings. First, successful DNA
typing results were obtained from nine of the ten psoas muscle samples.
Figure 1 shows the lumigraph produced during the hybridization step of this
research. Table 3 lists the results obtained from the computer assisted
lumigraph analysis.

Second, even though the samples were fresh tissue samples, the
quantity of DNA in each sample determined by the yield gel was
significantly low. Figure 2 shows the yield gel photo produced during this
research. Table 4 lists the results obtained from the yield gel interpretation.
Refer to Appendix B, “Determination of DNA quantity and quality from yield
gel”, to see the calculations performed during this step.

Third, the test gel indicated that there was a significant amount of
DNA in each sample to run an analytical gel. Only one sample produced no

results at all. Figure 3 shows a photo of the test gel. Table 5 lists the results

of the test gel analysis. Appendix D, “Determination of the completeness of
digestion from test gel”, contains the calculations performed during this step.
Fourth, the lumigraph was developed using a chemiluminescent

detection method. A successful lumigraph was produced in only 16 hours.

29

30

Table 3-Results of computed assisted lumigraph analysis

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample number DIZSll Probe Allele sizes (bp) D17S79 Probe Allele sizes (bp)

' 1 11.467 2.667
5.636 2.667
2 10.546 3.675
9.750 2.873
3 11.320 3.902
9.913 3.391
4 12.249 4.092
7.077 3.351

5 No results No results
6 10.999 3.885
7.047 3.286
7 11.129 3.814
10.328 3.636
8 11.994 3.330
11.268 2.824
9 9.793 3.622
7.310 3.362
10 11.920 3.848
6.900 3&9
Allelic control 15.952 4.074
7.582 3.593
Isolation control 12.540 3.818
7.293 3.433

 

 

 

Figure 1-Lumigraph developed with LumiPhos 480

 

32
Table 4-Results of yield gel interpretation

 

 

 

33

61'4”

 

 

Figure 2-Yield gel photo

34
Table 5-Results from test gel interpretation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lane number Sample number Amount of loading buffer

1 Pat Test Gel Standard (10 ul) ..............

2 1 40 ...........
3 2 20 ...........
4 3 20 ...........
5 4 45 45

6 5 20 ............
7 6 45 45

8 7 45 ............
9 8 45 90

10 9 45 ............
ll 10 45 90

12 IC 1/3 45 ............
13 RC 1/12 45 ............
14 Pat Test Gel Standard (20 ul) ..............

 

 

 

 

 

‘1‘... -- _

‘-

 

35

 

Figure 3-Test gel photo

CONCLUSIONS AND RECOMMENDATIONS

After a review of previous research, this project set out to determine if
useful DNA typing results could be obtained from psoas muscle tissue.
Although all but one of the samples provided usable results, further research
is necessary to determine if postmortem psoas muscle provides useable
results under extreme environmental conditions. The muscle tissue samples
analyzed in this research were pristine. Each sample was collected
approximately 24 hours after death and fi'ozen immediately. They were not
exposed to any harsh environmental conditions such as heat, ultraviolet
light, soil or humidity.

Another goal of this research was to determine if useable quantities of
good quality DNA could be extracted fi'om each sample. Only six of the ten
psoas muscle samples showed results on the yield gel. It was decided that
the procedure should proceed as normal with all ten samples. A possible
explanation for this is that the amount of DNA extracted from each sample
may be too small to visualize utilizing the yield gel but, there still may have
been a sufficient quantity to type. Fortunately, nine of the ten tissue
samples resulted in useful RFLP patterns. Previous research findings
indicate that the amount of degraded DNA correlated with the length of the

postmortem period. However, some DNA degradation was prominent after

36

37

short periods of time in some cases. Early degradation is usually the result of
high environmental temperature and/or infectious disease. Early
degradation is a possible explanation for why only one of the ten samples in
this research did not produce usable results. All the samples were frozen
immediately after collection. Therefore, good results would be expected from
all the muscle tissue samples.

The results obtained from the DNA yield gel indicated that all of the
psoas muscle samples yielded 2 5ug of DNA. The literature indicated that
greater than 50 ng of relatively undegraded DNA is necessary to produce
useful RFLP results. Therefore, the amount of DNA extracted fiom the
samples in the research exceeded the amount suggested by the to obtain
useful RFLP results.

As with all research projects, further research suggestions are made.
An alternative to RFLP typing is the polymerase chain reaction (PCR) typing
method. Previous research has indicated that successful PCR results have
been obtained fi'om samples whose DNA was degraded and yielded no RFLP
results. The application of the PCR technique has been successfully applied
to very small blood or semen stains, stains that contained degraded DNA and
even DNA extracted fi'om single hairs. An attempt could possibly be made to
use PCR typing to analyze the single psoas muscle that did not produce
useful RFLP results. The RFLP technique is extremely sensitive, reliable
and has been said to offer a higher power of discrimination than the PCR
technique.

Another important aspect of this research was the use of

38
chemiluminescent detection of the RFLP patterns. Both analysis time and

safety have been improved by the use of chemiluminescent labeled probes
verses the 32P-labeled radioactive probes. Analysis time utilizing the
radioactively labeled probes can take weeks to complete. The analysis time
using chemiluminescent labeled probes can be measured in hours. Only 16
hours were required to develop the lumigraph of the RFLP results produced
in this research.

Further research suggestions include performing a comparison study of
DNA extracted from different types of tissue, and perform a time line study
to determine how the results from fresh psoas muscle samples compare to

aged psoas muscle samples.

APPENDIX A

10.

11.

APPENDIX A

DNA ISOLATION FROM MUSCLE TISSUE

Label 2 sets of microcentrifuge tubes. The first set should be labeled
with the specimen number and the date. The second set of tubes are
labeled with just the specimen numbers.

Finely chop the muscle tissue (350 mg) in a disposable petri dish with
saline, using a fresh scalpel blade.

Add 1.0 ml of the diced muscle tissue to a labeled centrifuge tube.

The LIFEPRINT CONTROL should be processed with the samples
beginning at step 5.

Add 1.0 ml Cell Lysis Bufi'er. Cap and vortez 1 minute. Visually
inspect specimens to ensure even suspension. Manual resuspension
with pipeters and tips may be necessary to break up any clumps.

Centrifuge on high speed for 2 minutes. Decant supernatant. Blot
each tube on kimwipe.

Repeat steps 5 and 6 two (2) times. An additional wash may be
performed if red cells remain. (Heme will inhibit the reaction).

Add 1.0 ml Protein Lysis buffer. Let samples sit for approximately 20
minutes. Vortex to resuspend pellet, making sure there are no clumps
of cells.

Centrifuge at high speed for 2 minutes. Decant supernatant. Blot.
PLACE SAMPLES ON ICE.

Immediately before use, prepare a master mix of 225 111 Protein Lysis
Buffer and 25 ul PRO-K per sample, PLUS one extra aliquot
compensate for pipetting tolerances. Mastermix MUST BE KEPT ON
ICE AT ALL TIMES.

Processing one sample at a time: Add 250 ul of Mastermix. Pipet up

and down to THOROUGHLY resuspend pellet. MIX WELL. Place
tube in 65° C heat block.

39

12.

13.

40

NOTE: It is very important that the cell pellet be completely
resuspended when Mastermix is added. Do not rely on vortexing
alone.

Begin two hour incubation after last tube is added to heat block.
Vortex every 15-20 minutes to INSURE nuclear pellet is resuspended.

Vortex vigorously for 30 seconds following complete incubation.
Microcentrifuge for 2 minutes.

NOTE: The procedure may be stopped at this point and samples
may be stored at 4° C.

APPENDIX B

APPENDIX B

DETERINATION OF DNA QUANTITY AND QUALITY FROM YIELD
GEL

1. Vortex samples for 15 seconds. Microcentriguge briefly to bring
ontents to bottom of tube. Incubate 5 minutes at 65° C.

2. Microcentrifuge briefly to bring contents to bottom of tube.

3. Add 2.4 ul DNA sample and 9.6 ul Yield Gel Loading Bufl’er to second
set of labeled microcentrifuge tubes. Store unused samples at 4° C.

4. Incubate all samples, Visual Marker, Calibration Control, and Yield
Calibrators for 5 minutes at 65° C. Vortex briefly. Microcentrifuge all
tubes briefly.

5. Load first row of Yield Gel in the following manner:

1. 30 ng/ul Calibrator (10 ul)
2. 20 ng/ul Calibrator (10 ul)
3. 15 ng/ul Calibrator (10 ul)
4. 10 ng/ul Calibrator (10 ul)
5. 5 ng/ul Calibrator (10 ul)
6. Calibration Control (10 ul)
7. Sample # (10 ul)
8. . (10 ul)
9. (10 ul)

(10 ul)
. . (10 ul)
17. Sample # (10 ul)

6. Subsequent rows in the SAME gel may be run minus the calibrators.

7. Electrophorese between 50-100 volts for 45-60 minutes. Photograph
under UV on low setting.

INTERPRETATION OF YIELD GEL:

1. Estimate the quantity (ng) of high molecular weight DNA loaded for
each sample be comparing the band size and the intensity of the
unknowns with those of the Yield Calibrators. Since 10 ul of each

41

42

calibrator was loaded, the calibrators represent 300 ng, 200 ng, 150 ng,
100 nmg and 50 ng of DNA respectively. The Calibration Control
should compare to the 100 ng standard. Significant variation of the
Calibration Control should be noted and investigated. Call Lifecodes
to verify that there are no problems with the control, and/or run a new
yield gel to confirm the reuslts.

If samples contain significantly less than 50 ng of DNA, an additional
aliquot of sample shoud be processed and combined. Reanalyze on
another yield gel.

For samples containing significantly greater than 300 ng of DNA,
dilute with 250 ul of Protein Lysis Buffer. Vortex to mix. Reanalyze
sample on another yield gel.

High molecular weight DNA should appear as a tight band slightly
above the uppermost band of the Visual Marker. If the sample shows
“trailing”, use only the region above the upper band of the Visual
Marker for quantifying high molecular weight DNA. If the DNA shows
significant levels of degradation, a fresh sample may be required.

Calculate the concentration of DNA for each sample by dividing the
quantity of DNA (ng) by the volume (ul) of the sample aliquot used in
the yield gel. For example:

Sample compares to 100 ng calibrator (10 ng/ul)
100 ng DNA / 2 ul sample = 50 ng/ul

The LIFEPRINT Control should have a concentration of approximately
50 ng/ul. Significant variation may indicate a procedural error. A
yellow discoloration of the original pellet may indicate age and/or
impoper storage. Call Lifecodes to verify that there are no problems
with the control, and/or run a new yield gel to confirm results.

The concentration of DNA in each sample is required for subsequent
restriction digestion. Record results and reagents on Yield Gel
Worksheet.

APPENDIX C

APPENDIX C

RESTRICTION DIGESTION OF ISOLATED DNA

Label 3 sets of microcentrifuge tubes. Label one set with sample
number and the date. Label the two sets with sample numbers only.

Using the table below, mix together in a labeled set of microcentrifuge
tubes, the appropriate amount of sample and distilled H20.
Refi'igerate genomic DNA samples after using.

Table 6-Preparation for restriction digestion

 

 

 

 

 

 

 

 

Concentration Amount of distilled H20 Amount of Sample
of Samples DNA

250 ng/ul 180 ul 20 ul

150 ng/ul 166 ul 34 u]!

100 ng/ul 150 ul 50 ul

75 ng/ul 133 ul 67 ul

50 ng/ul 100 ul 100 ul

25 ng/ul --------- 200 ul

 

 

 

 

Prepare a master mix of 154 ul Nuclease-free H20, 40 ul Digestion
Buffer, 4 ul Spermidine, and 2 ul Pst I Enzyme for each sample, PLUS
one extra aliquot to compensate for pipetting tolerances. Mix well by
inversion. KEEP MASTER MIX ON ICE.

NOTE: Remove Pst I Enzyme from freezer immediately prior to
use and place on ice. Prepare master mix immediatley before use and
place on ice. Return unused enzyme to fi'eezer immediately.

Add 200 ul of master mix to each tube. Gently flick tube to mix.

Incubate at 37° C for 2 hours. After 30 minutes has elapsed, gently
flick each tube. Microcentrifuge briefly at end of incubation.

43

10.

11.

12.

13.

14.

44

NOTE: The procedure may be stopped at this point and samples
stored at -20° C. Rewarm samples for 5 minutes at 65° C

Add 200 ul of lithium chloride Conditioning Salt. Vortex 5 seconds.
Place on ice for 13 minutes.

Microcentrifuge for 10 minutes. Transfer supernatants to a fully
labeled set of microcentrifuge tubes. Avoid transferring any of the
precipitated protein and other debris. The pellet is often difficult to
visualize.

Add 1.0 ml room temperature 95% Ethanol. Invert to mix. Incubate
at room temperature for 20-30 minutes.

Microcentrifuge for 30 minutes to pellet DNA. Decant supernatant.
Blot.

Add 1.0 m1 room temperature 70% Ethanol. Vortex well for 3 minutes.

Microcentrifuge for 30 minutes to pellet DNA. Carefully decant
supernatant. (Cell pellet does not adhere tightly to walls of tube).
Blot.

Dry pellet with Speed-Vac for 15-30 minutes. (This may be shortened
to 10 minutes if the heat is turned on; however, excessive drying will
make the pellet hared to resuspend). The procedure may be stopped at
this point and samples stored at -20° C.

Add 51 ul of Nuclease-free H20. Incubate at 65° C for 3-5 minutes.
Vortex. Re-incubate at 65° C for 5 minutes.

Vortex to completely resuspend. Microcentrifuge briefly to bring
contents to bottom of the tube.

NOTE: The procedure may be stopped at this point and samples
stored at -20° C. (If storedz) Reheat samples for 5 minutes at 65° C.

APPENDIX D

APPENDIX D

DETERMINATION OF THE COMPLETENESS OF DIGESTION FROM
TEST GEL

1. Refer to Appendix F, “Procedure for Preparation of Agarose Gels,” for
the preparation of the test gel(s).

2. If the restricted DNA has been stored at -20° C, heat samples for 5
minutes at 65° C, vortex, and microcentrifuge briefly. All 6 ul of
restricted DNA samples and controls to 6 ul of Loading Buffer in the
last set of labeled microcentrifuge tubes.

NOTE: Store the restricted DNA sampes at -20° C immediately
after use.

3. Incubate DNA/Loading Buffer mixtrues, Visual Marker and Pst Test
Gel Standard for 5 minutes at 65° C.

4. Vortex. Microcentrifuge all tubes briefly to bring contents to bottoms
of the tubes.

5. Load first row of Test Gel in the following manner:

1. Visual Marker (10 ul)

2. Pst Test Gel Standard (10 ul)

3. LIFEPRINT Control (10 ul)

4. GENOMIC DIGESTION Control (10 ul)

5. Sample (10 ul)

6. Sample (10 ul)

. (10 ul)

. . (10 111)

(up to) 17. Pst Test Gel Standard (20 11])

Subsequent row in the same gel may be run minus the standards.

6. Electrophorese between 50-100 volts for one hour. Photograph under
UV ( “Low” setting).

7. Interpretation of test gel:

a. Determine for each sample if the Pst I digestion is complete by

45

46

comparing its pattern with the Pst Test Gel Standars. The digestion
pattern should be a uniform smear with a well defined starting point
slightly above the uppermost band of the Visual Marker.

b. Compare the smear intensity of the samples to the standards to
insure that the appropriate amount of DNA will be loaded onto the
Analyical Gel. The fluorescent intensities of the Pst Test Gel Standard
are equivalent to 500 ng and 1 ug of digested DNA. The 5 ul of sample
used on the test gel should contain between 500 ng and 1 ug of DNA.

c. The DIGESTION Control should contain approximately 500 ng
of Pst I digested DNA. This control should reproducibly cut to
completion under the conditions of this protocol with Pst I Enzyme and
is a control of enzyme reliability. If the digestion control has not
completely restricted, the reactivity of the enzyme should be confirmed
through Lifecodes. Redigestion of all samples may be necessary.

d. Significant variation in concentration must be corrected before
samples are loaded onto the Analytical Gel. If fluorescence of any
sample is significantly less than the 500 ng standard, an additional 5
ug of DNA must be digested. If the fluorescent intensity of a sample is
significantly greater thatn the 1 ug standard, the sample should be
diluted with appropriate amount of Loading Buffer.

If the Pst restriction digestion is complete, add 45 ul Loading Buffer to
each sample. This amount may vary due to DNA concentration in the
restriciton. Proceed to Analytical Gel section.

If the restriction is not complete, proceed as follows:

a. Remove sample to be “re-digested” fiom the fi'eezer and add 5 ul
Digestion Buffer and 2 ul Pst I Enzyme. Incubate 2 hours at
65°C After 1 hour has elapsed, flick tubes to insure suspension
of enzyme.

b. Add 2 ul Conditioning Salt and 100 ul 95% Ethanol. Invert
tube to mix. Incubate 20-30 minutes at room temperature.

c. Microcentrifuge 20 minutes to pellet DNA. Decant supernatant.
Blot. (Note: Pellet does not adhere tightly to wall of tube.)

(1. Add 1.0 ml room temperature 70% Ethanol. Vortex well.

e. Microcentrifuge 10-20 minutes to pellet DNA. Carefully decant
supernatant. Blot.

47
Dry pellet with Speed-Vac for 5-10 minutes.

Add 50 ul Nuclease-free H20.

Repeat test gel procedure and interpretation. If restriciton
digestion is still incomplete, discard sample.

APPENDIX E

APPENDIX E

ANALYTICAL GEL ELECTROPHOESIS

Prepare as many Analytical Gels as needed to run completed samples
and controls.

NOTE: Refer to Appendix F, “Procedure for Preparation of
Agarose Gels”, for the preparation and assembly of analytical gels.

Remove samples and controls fiom freezer and thaw.

Microcentrifuge briefly all samples , Allelic Control, Sizing standards,
and other controls.

Incubate 5 minutes at 65° C. Vortex. Microcentrifuge for 5 minutes.

Load 10 ul of sizing standard and 20 ul of each sample into an
analytical gel. The following pattern is recommended:

Lane Sample

1. Sizing Standard
2. Sample 1

3. Sample 2

4. Sample 3

5. Sample 4

6. Sample 5

7. Sample 6

8. Sample 7

9. Sample 8

10. Sample 9

11. Sample 10

12. Sizing standard
13. Allelic Control

NOTE: The Allelic Control MUST be run on every plate. The
LIFEPRINT Control and /or Digestion Control must be run with each
set of samples processed together.

Electrophorese at 14.5 volts acroos the top gel for 65 hours. If
electrophoresis will take place for a longer or shorter period, adjust

48

49

voltage proportaionatly. After the first hour has elapsed, turn on the
circulating pumps.

NOTE: If running more than on gel box, be certain to run the
same number of gels in each box.

APPENDIX F

APPENDIX F

PROCEDURE FOR PREPARATION OF AGAROSE GELS

Notes: a) Yield gels and test gels can be prepared in 12 x 15 cm or
12 x 27 cm gel trays. Analytical gels are always
prepared in 12 x 27 cm gel trays. All the gels utilize 0.6%
agarose.

b) The instructions below are for preparing one 12 x 15 cm
(100 ml) or one 12 x 27 (200 ml) gel. The amount of
agarose should be adjusted for additional gels.

c) The Gel Buffer concentrate must be diluted 1:40 with
distilled water before use.

1. Determine the number of gels needed. Listed below are the amounts
of agarose, Gel Bufl‘er and ethidium bromide required per gel.

Yield/Test Gels Analytical Gels

Small (100 ml) Large (200 ml) Large (200 ml)
Amount Agarose 0.6 g 1.2g 1.2g
Amount Gel Bufi'er 100ml 100 ml 200 ml
Ethidium Bromide 5ul 10 ul -------

2. Prepare gel trays by taping the open ends.

3. Measure diluted Gel Bufl‘er in the graduated cylinder and pour into

an Erlenmeyer flask large enough to hold at least 2 times the volume
of the buffer to be added.

4. Weigh agarose, add to flask, and swirl gently. Heat on a heated stir
plate, until all agarose is dissolved. Make sure all crystals are

dissolved before proceeding.

5. After heating, check volume of the solution in a graduated cylinder.
Add distilled water to replace any volume lost in heating.

6. For yield and test gels, return solution to flask. Add 5 ul ethidium
bromide per 100 ml of gel. Swirl contents to mix.

50

51

Pour molten agarose into center of gel tray. Be sure tray is on a level
surface. Remove any bubbles with tip of transfer pipette. Place combs
into position. One to four rows of wells may be used for yield gels and
test gels to accommodate more samples.

APPENDIX G

APPENDIX G

PREPARATION OF TRANSFER WORKING SOLUTIONS

 

DENATURING SOLUTION 10 L

NaCl 584 g

NaOH 200 g

Distilled H20 10 L
NEUTRALIZATION SOLUTION 4 L 10 L
NaCl 584 g 1460 g
Tris 39 g 97 g
Tris HCL 661 g 264 g

Distilled H20 4 L 10 L

52

APPENDIX H

APPENDIX H

Southern Transfer of DNA onto nylon membrane

Stack gel trays on top of each other in a rectangular tray. Place an
empty tray on top.

Carefully pour in enough Denaturing Solution to completely submerge
gels (approximately 750 mls/gel). Let sit for 20-30 minutes.

Discard Denaturing Solution. Rinse gels carefully two times with
distilled water to remove excess Denaturing solution.

Add enough Neutralization Solution to completely submerge gels
(approximately 750 mls/gel.) Let set 20-30 minutes.

Prepare nylon membranes:

a. For each gel, label a membrane with a pencil in lower left corner
with gel #, sample #8 and technologists' initials, and set up
date.

b. Add 250 mls distilled water to a tray and carefully immerse
membranes so that they wet evenly.

c. Replace water with 250 mls of Neutralization solution.
Submerge membranes and let soak until ready for use.

Prepare sponges:

Rinse sponges thoroughly with tap water. Wring dry.

Rinse with distilled water. Wring dry.

Soak in Neutralization solution. Wring dry.

Saturate sponges with fresh Neutralization solution. Let stand
until needed.

999‘!”

Onto Plexiglas bench shield, stack for each gel to be transferred, three
saturated sponges and two sheets blotting paper pre-wet with
neutralization solution. Remove air bubbles between the sponges by
rolling gently with a disposable pipette.

Carefully remove gel trays fi'om Neutralization solution and slide gel
from tray onto the top of the blotting paper. Carefully smooth gels to
remove excess buffer from tops. Remove air bubbles between gel and
blotting paper.

53

10.

11.

12.

13.

14.

15.

16.

17.

54

Place pre-wet, labeled nylon membrane on top of the gel. The labeled
side should be facing upwards, and the writing should be on the lower
edge of the gel. Remove all air bubbles and ensure complete contact by
gently rolling a disposable pipette over the membrane.

Pre-wet two sheets of blotting paper with neutralization solution and
carefully place on top of the nylon membrane. Do not reposition
blotting paper without checking for air bubbles that may have been
introduced between the nylon membrane and the gel. Gently roll a
pipette over the blotter paper to remove any bubbles.

Stack two inches of paper towels and a gel box lid or Plexiglas weight
on top of the gel. Ensure uniform contact and even weight
distribution. Make sure no paper towels overlap onto sponges or in
buffer. They should only touch the blotter paper on top of the
membranes.

Allow transfer to proceed for 3-4 hours. After one hour has elapsed,
replace the wet papertowels with dry ones.

Disassemble transfers one at at time.

a. Carefully peel nylon membrane away fi'om gel. Place membrane
in approximately 500 ml of 1:5 dilution of neutralization
solution and water.

b. With a gloved hand, gently rub the side of the membrane that
was in contact with the gel to remove any residual agarose.
Turn the membrane over and repeat on the other side.

c. Place membranes on a sheet of filter paper and allow to air dry
until membranes are no longer glossy.

Repeat step 13 for each membrane. The same solution may be reused.

Sandwich the membranes between 12 X 28 cm sheets of filter paper in
a stack. Do not stack more than 10 membranes. Bake at 65-80° C for
one hour or until completely dry.

Place each membrane in a 302 nm UV transilluminator with DNA side
facing the UV source (the writing should be facing upwards). Expose
to UV for 90 seconds.

Place membranes into heat sealable bags until ready for hybridization.
Membranes may be stored indefinitely.

APPENDIX I

APPENDIX I

HYBRIDIZATION OF NYLON MEMBRANES

Note: It is recommended that 1-5 membranes be processed together per vial
of probe. Slightly more membranes may be processed with a small decrease
of probe signal resulting. Membranes must be handled with gloves and
should only be touched on the edges.

1.

Prepare 1X Quick-Light Buffer, wash I and II as follows:
(prepare QUICK-LIGHT on day of use only)

1X QUICK-LIGHT Buffer 900 ml deO
100 ml Quick-Light Bufl'er

Wash I 40 ml Wash Component A
250 ml Wash Component B
710 ml deO

Wash II 4 m1 Wash Component A
50 ml Wash Component B
946 ml deO

To process 5 membranes, place Lumi-Phos 480 at room temperature,
and incubate the following reagents at 55° C for 90 minutes prior to,
use:

75 ml Hybridization solution

2,000 ml Wash I

2,000 ml Wash II

Note: To hybridize fewer membranes, use 20-25 ml hybidization
solution for one membrane (300 cm2 membrane) and 10-15 ml for each
additional membrane. Use up to 100 ml of all other solutions per
membrane. It is important to add sufficient solution to have all the
membranes completely immersed.

Trim membranes to approximately 25 cm in length, sort membranes,
and place in appropriate sized clean container containing 500 ml of
Wash I at 55° C. Using forceps, separate each membrane to allow
complete wetting. Cover container with leakproof lid and shake at 55°
C for at least 10 minutes.

55

10.

11.

12.

13.

14.

15.

56

During step 3, combine Probe Solution with 75 ml of Hybridization
solution pre-heated to 55° C. Screw the cap on tightly, secure the cap
with parafilm and incubate at 55° C for 10-20 minutes before use.

Pour out the Wash I solution and all 75 ml of the Probe Solution pre-
heated to 55° C. Cover and shake at 55° C for 10-20 minutes.

Note: Incubations longer than 30 minutes may result in an increase
in background.

Pour out the Probe Solution and add 1 liter of Wash I pre-heated to 55°
C. Cover and shake at 55° C for at least 10 minutes.

Pour off the Wash Solution I and repeat step 6.

Pour off the second Wash Solution I and add 1 liter of Wash Solution
II pre-heated to 55° C. Cover and shake at 55° C for at least 10
minutes.

Pour off the Wash Solution II and repeat step 8.
Pour off the second Wash Solution 11.

Add 500 ml of 1X QUICK-LIGHT Bufi'er. Cover and shake the
container vigorously. Using forceps, separate each membrane to allow
complete wetting.

Pour off 1X QUICK-LIGHT Buffer and repeat step 11 three times.

Allow excess QUICK-LIGHT Bufi'er to drain. Position up to three
rinsed membranes on an open development folder. Spray each
membrane evenly with Lumi-Phos 480, using the spray applicator
provided.

Close development folder over the sprayed membranes and remove air
bubbles and excess Lumi-Phos 480 by firmly wiping the closed folder
with a papertowel. Seal the folder with an impulse heat sealer.
Inspect seals to ensure that the Lumi-Phos will not leak. (The

folders and membrane should be trimmed so that they are
approximately 25 cm in length, otherwise they will not fit into
cassettes.)

Wipe the surface of the folder dry with a paper towel before inserting it
into a X-ray cassette.

57
16. Expose film overnight at room temperature.

17. Develop film and review resulting Lumigraphs.

APPENDIX J

10.

11.

APPENDIX J

REAGANTS

Reagents from Lifecodes, Inc.

Cell Lysis Buffer: Contains sucrose, magnesium chloride, and Triton
X-100 in Tris Buffer. Store at 2-8° C.

Protein Lysis Buffer: Contains sodium EDTA and sodium chloride in
Tris buffer. Store at 2-8° C.

Pro-K: Contains proteinase K in Tris buffer. Store at -20° C.

Gel Buffer: Gel Buffer contains sodium EDTA in Tris-acetate buffer as
a 40X concentration. Dilute 1:40 in nanopure water to prepare
Working Gel buffer. Store at room temperature.

Visual Marker: Visual Marker contains Hind III digested lambda
DNA in loading buffer. Store at -20° C.

Yield Calibrator Set: Yield Calibrator Set includes calibrators
containing lambda DNA in loading buffer at concentration of 30 ng/ul,
20 ng/ul, 15 ng/ul, 10 ng/ul and 5 ng/ul.

Calibration Control: Calibration Control contains 10 ng/ul high
molecular weight human DNA in loading buffer. Store at 28° C.

LIFEPRINT Control: LIFEPRINT Control contains 2.5 X 10
lyophilizied K562 human cells per vial. Store at 2-8 ° C.

Yield Gel Loading Buffer: Yield Gel Loading Bufl'er contains Ficoll
400, bromophenol blue, sodium EDTA, and SDS in Tris-acetate buffer
as a 1.25X concentration. Store at room temperature.

Ethidium Bromide: Ethidium Bromide contains 10 mg/ml ethidium
bromide. Store at room temperature. Mutagen

Conditioning Salt: Conditioning salt contains lithium chloride. Store
at room temperature.

58

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

59

Pst I Enzyme: Pst I Enzyme contains 50 U/ul Pst I, Tris-hydrochloric
acid, sodium chloride, EDTA, bovine serum albumin, and B-
mercaptoethanol in Tris buffer. Store at -20° C.

Digestion Buffer: Digestion Buffer contains sodium chloride,
magnesium chloride, bovine serum albumin and B-mercaptoethanol in
Tris buffer. Store at -20° C.

Spermidine: Spermidine contains a diluted aqueous solution of
Spermidine. Store at -20° C.

Nuclease-flee H20: Nuclease-free H20 contains autoclaved, purified
H20. Store at room temperature.

Hybing Solution: Hybing Solution contains polyethylene glycol,
sodium chloride, sodium phosphate, EDTA, sodium dodecyl sulfate,
sodium heparin and herring testes DNA. Store at 28° C.

Wash Component A (SSPE): Wash Component A contains sodium
chloride, sodium phosphate and EDTA as a 25X concentration.
Contains Sodium Azide. Store at room temperature.

Wash component B (SDS): Wash Component B contains sodium lauryl
sulfate (SDS) as 20% solution. Contains Sodium Azide. Store at
room temperature.

Loading Bufi'er: Loading Buffer contains Ficoll 400, Bromophenol
blue, and sodium EDTA in Tris-acetate buffer. Store at room
temperature.

Digestion Control: Digestion Control contains 100 ng/ul high
molecular weight K562 human DNA and EDTA in Tris buffer. Store
at 2-8° C.

Pst Test Gel Standard: Pst Test Gel Standard contains Hind III
digested lambda DNA in loading buffer. Store at -20° C.

Allelic Control: Allelic Control contains 50 ng/ul of Pst I
digested K562 human DNA in Loading Bufi'er. Store at -20° C.

Sizing Standards: Sizing Standards contains DNA fi'agments of
various lengths originating fi'om PhiX174, M13, Lambda and T7 phage
in Loading Buffer. Store at -20° C.

24.

25.

26.

27.

28.

60

Combination Standards: Combination Standards contains Adenovirus
digested with Kpn I enzyme and the Sizing Standards in Loading
Buffer. Store at -20° C.

Lumi-Phos 480: Store at 28° C. Avoid Aerosol

I.D. Na Agarose: Store in dry location at room temperature. Use for
analytical gels.

QUICK-LIGHT Buffer: Store at room temperature. Contains
Sodium Azide.

Probe Solution 1 (D12S11/D17S79): LIFEPRINT Probe Set I contains
the enzyme labeled DNA probes D12Sll and D17 S7 9 plus enzyme
labeled phage DNA ( PhiX17 4, M13, Lambda, T7) for the molecular
weight markers. The chemiluminescent substrate (Lumi-Phos 480) is
added to react to the hybridized probe. Store at 2-8° C.

Reagent other than Lifecodes

Agarose, Seakem LEzFMC Bioproducts, Cat. #50003. Store in dry
location at room temperature. Use for yield and test gels.

Sodium chloride: SP Cat #7581-12 NY. Store at room temperature.
Sodium hydroxide: SP Cat #7 708-25 NY. Store at room temperature.
Trizma base (Tris): Sigma Cat. #T-1503. Store at room temperature.

Trizma-HCL (Tris-HCL): Sigma Cat. #T-3253. Store at room
temperature.

Ethanol, 95%: AAPER Alcohol and Chemical, order through Sparrow
Pharmacy. Flammable. Store in flame-proof chemical storage cabinet
at room temperature.

APPENDIX K

APPENDIX K

COMPUTER-ASSISTED LUMIGRAPH ANALYSIS SIZING PROGRAM

1.

Call the DNA directory to your PC. After the C:\> prompt appears,
type cd\DNAPHI91.

Begin the DNA sizing program. After the C:\DNAPH191> type: D.

“LIFEPRINT (tm) SIZING PROGRAM FOR PATERNITY” will appear
on the screen. Press the digitizer button once to insure proper hook-
up. If a beeping sound is not heard, make sure connections are
properly in place and start program again.

The computer will prompt you for optional information. Press “enter”
to skip or type today’s date, press “enter”; type date gel was run, press
“enter”; type initials of person who ran the gel, press “enter.” After
pressing enter the last time, a question appears on the screen. Should
you find that something was entered incorrectly, move the cursor to “2
no.” Otherwise press “enter” to proceed.

The program requires you to input a gel number, which will be part of
the name of the file under which the data are stored. Type gel
number, without using a hyphen, and press “enter.”

The other part of the file name is the initials of the person who is
sizing the lumigraph. Type initials, press “enter.”

The next screen will present the name of the file you are currently
creating. If any lumigraphs have previously been sized under the
same file name, you have a choice of whether you:

a. want the sizing you are currently working on to overwrite the
previously named file or,

b. add what you are currently sizing to the existing file, or

c. rename the file that you are currently creating by reentering

the gel number and your initials.

Choose the appropriate option, and press “enter.” If there are no
existing files with the same name, simply press “enter.”

61

10.

11.

12.

62

The next screen requests that you enter the number of paternity cases
on the lumigraph. Generally, paternity cases are ordered so that
standard size marker lanes flank each case. The program
automatically takes this ordering system into account, and will,
therefore, expect the number of standard size marker lanes that need
to be sized, to be “one, plus the number of paternity cases on the
lumigraph”. Type the number of paternity cases, and press “enter.”

For the first paternity case on the lumigraph (the left most), choose the
option that accurately describes the paternity case. Move the cursor to
the appropriate number and press “enter.”

NOTE: If you choose “4. none of the above," you will be expected to
first enter the number of lanes of the gel that the case occupies. This
number should include mixture lanes, if any. Then, the case number
should be specified. After typing the case number and pressing
“enter," each lane is displayed on the monitor and it is necessary for
you to type the suffix for each lane, in order, as it appears on the
lumigraph. Use -10 for the mothers, -20 for children, -30 for alleged
fathers, -X1 for first mix, -X2 for second mix, etc. After typing the
suffix for the first sample, press “enter” and repeat for each subsequent
sample lane for the case.

After each paternity case is described, the race of the mother and
alleged father(s) may be entered, or for choice number “4”, you are
given the option to enter race for each sample lane. Move the cursor to
the appropriate choice, and press “enter.” (Note: enter “5-unknown” for
each.)

The next screen gives you the option to edit information regarding case
sample numbers and race. In addition, if you incorrectly entered too
many samples, such that greater than 17 lanes including the control
lane are recorded, a statement will appear on the top of the screen to
inform you, and you must reenter the number of cases.

After reviewing the editing screen, and having determined that all
sample and race information is accurate, you can proceed by
answering “yes” to the question “has you sample number and race
information been entered correctly?”

You must now make a choice of the probe, whose alleles (represented
by bands on the lumigraph) for each sample lane will be what you are
going to size. Move the cursor to the appropriate choice, and press
"enter."

13.

63

You can recheck that you have entered the probe information
accurately. Move the cursor to “yes” when asked the question “Is the
probe entered correctly?”, and press “enter."

On the next screen are numbers corresponding to the size standards.
To determine what standards you need to include, first look for the
highest allele and lowest allele of the probe currently being sized (in
all the sample lanes). Then choose a standard band, which is higher
than the highest allele, as the standard with the greatest size.
Choose a range that includes at least seven size standard bands, but

no greater than 25 bands, to get an accurate determination of goodness
of fit.

Type the number of the highest size standard band after the statement
“enter the number of the standard with largest size [?].” '

Next, choose a size standard band lower than the lowest allele. Type
the number corresponding to the lowest size standard band after the
statement “enter the number of standard with lowest size [?].”

If you entered either or both of these numbers incorrectly, you may
reenter them. Choose the appropriate answer to the question “Are the
standards correct?”, and press “enter.”

Sizing the standards:

The proper order for sizing these bands is to start with the band of
greatest size and proceed, sizing each band in numeric order until you
reach the one with the smallest size.

1. When “ENTER STANDARD # ” appears on the screen,
center the cross-hairs of the mouse on the appropriate band (the
largest standard you have chosen to size) in the standard lane,
and press the button on the mouse.

2. Complete the sizing of all the bands in the lane. The screen

will then provide information in columns, under the heading
“LANE NO 1.”

Below is a list of the column descriptions:

a. The number-from highest to lowest- associated with the
respective size standard band.

b. The Hi-pad’s corresponding number related to the point
at which mouse button was pressed.

64

6. Expected size of the standard band.
d. Observed size of the standard band.
e. Error associated with each observed sizing.

The goodness of fit is displayed below the numbers in the
numbers in the columns. The goodness of fit must be below 2 in
order to proceed. If the goodness of fit is greater than 2, after
the question “Go on?”, move cursor to “2 no”, and press “enter”.
You can then resize the same standard lane.

Once you get a goodness of fit less than 2, move cursor to “1 yes,"
and press “enter”. Repeat the sizing procedure for the second
standard lane. Depending on how many cases are on the
lumigraph, you may then have to size standard lane #3,
standard #4, etc. Once you have been successful in obtaining a
goodness of fit below 2 for each standard lane, you may proceed
to sizing of the sample lanes.

D. Sizing the Alleles in the Sample Lanes:

1.

For each sample lane, the screen displays a table with the
following headline: “Lane No DIGITIZE sample # ”.
The lane number corresponds to one of seventeen lanes (starting
with the left most lane of the lumigraph = Lane No l, and the
rightmost lane = Lane No 17) in which the sample resides. The
sample number plus the suffix indicating whether it is the
mother, child, alleged father, or mixture lane.

 

Size the sample lane:

a. If the sample lane contains only one band (homozygous)
for the probe system, center the mouse on the band, and
hit the button on the mouse one time.

b. If the sample lane contains two bands (heterozygous) or
more than two bands (in the case of a mixture lane), size
each band once, starting with the greatest size, and
proceeding in order of the lowest band. Center the cross-
hairs of the mouse on the band, and press the button on
the mouse.

Once you finish sizing the lowest band, you must place the cross-
hairs of the mouse within 1 inch of the left hand side of the
digitizer and press the button. This will indicate that sizing of
the lane is complete, and allows you to proceed to the next

65

sample lane. Record results on worksheet before proceeding to
next allele.

4. Repeat the same procedure for all sample lanes until you have
completed the last sample on the lumigraph, which should
always be the control lane.

5. The sizes of the alleles that are determined for the control lane
must be within 2% variation compared to the expected sizes,
otherwise repeat the sizing, since this may be an indication that
sizing of standard bands was done inaccurately.

If the control can not be sized within the expected range, the
plate must be re-run. No sizings are to be used from the plate
where the standards may be incorrect.

6. After you have finished sizing each lane, the sizing information
for that lane appears on the screen. The following is a

description of the information contained within the columns on
this table:

sample number.

allele number for each respective sample.

race of sample.

goodness of fit of left flanking size standard lane.
goodness of fit of right flanking size standard lane.
size of the allele (represented by band that was sized).

939993.”?

When asked the question “Are you finished?”, move the cursor to the
appropriate choice, and press “enter."

After completing the sizing of all samples, if your lumigraph contains
more than one probe system, you may size the alleles of the second
probe system. By answering “no” to the question “Are you finished”,
you will be brought back to the same screen that you have seen before
regarding the information about probe systems, and asked to enter the
second probe system. You then choose the number that corresponds to
the second probe system. Repeat all necessary steps in order to do the
sizing of all lanes for the second probe. If there is not another probe
system for data analysis, answer “yes” to the above question.

APPENDIX L

APPENDIX L

Function of select reagents and controls

Agarose

Alcohol (7 0%)

Alcohol (100%)

Allelic Control

Bovine Serum Albumin (BSA)

Calibration Control

Ethylenediamine Tetraacetic Acid (EDTA)

Ethidium Bromide (EtBr)

66

Medium used to separate
DNAfi'agments according to
size.

Washes salts and buffers
away; returns layer of
hydration to the DNA.

Precipitates the DNA and
removes protein and salt.

Removes the layer of hydration
of DNA.

Human genomic control that
must be run on every
analytical gel. The allelic
control is sized by the sizing
program and must fall within a
2% size range of the expected
kilobase size stated by
Lifecodes.

Enzyme stabalizer.

Run, at least, in one lane on
every yield gel. The Calibration
Control compares to the 100 ng
standard. Failure of this
control may indicate a need to
rerun yield gel(s).

Chelates Mg so the nucleases
cannot chew apart DNA.

Used as a fluorescent tracking
dye and for quntitation of DNA.
Interchelates between stacked
bases of DNA. Excited at 300

67

Genomic Digestion Control

Herring Sperm

Hybridization Solution

Lifeprint Control

Proteinase K

Spermine

Sodium Chloride

SDS

nm (UV light) quantitatively
absorbed by DNA.

Contains approximately 500 ng
of Pst I digested DNA. This
control is a check on enzyme
reliability. This control is
processed with every batch of
restriction samples.

Blocks unrelated repetitive
DNA probe and target; also
used in labeling as a seed to
precipitate DNA.

PEG, SDS, SSPE, H20

Should have a concentration of
approximately 50 ng/ul. This
control is started at the
extraction and run through the
yield gel, test gel and analytical
gel.

Proteolytic enzyme that
degrades DNAse and other
proteins into their constiuent
amino acids.

Binds unbound dNTP’s in
probe makeing; can be
added at RE time to tie
up negatively charged
contaminants.

Keeps DNA rigid so that
proteinase K can react on it.

Sodium Dodecyl Sulfate;
Protein denaturant; lyses
cell wall; denatures enzymes;
dissociate nucleic acid from
proteins-DNA complex;

SSPE

Tris

68

solubilizes membranes of
nuclei.

Sodium Chloride (NaCl) &
Sodium Dihydrogen Phosphate
(NaH2P04) & Sodium EDTA
Buffer used in hybridization
and blot stripping.

Buffer used to maintain pH.

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69

70

Honma, M., Yoskii, T., Ishiyama, I., Mitani, K., Kominami, R., and
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Kirby, LT. (1992). DNA fingerprinting an introduction. New York: W.H.
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71

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