3.4? . . 1 .II If... Cl 5 .y.!r::u!..flrll P so it: . u v) ‘frb‘O‘l‘ IV . Efimsci . .- .l... 9.30.. s. 03.1.71: 3 x l . 45.9: p .Kvfl't...‘ . t... _ . $31! $.13 it ran.” .ueflummhtvur: i.).vxl:(n 4“! In." fwfilIOl’n. .01vrv?l .11!“ n‘ v . 9.447(094‘” A l‘ . 5. . 1i .x. :7. £43....“ fa? . kaiifi i’ i \ 7‘ “JO ||\||\\U|HHIIHHHHHIHWWilli \[ ltliliilll 3 1293 02060 This is to certify that the thesis entitled DEVELOPMENT OF A MICROSATELLITE MARKER PANEL FOR GENO‘TYPING‘MICHIGAN WHITE-TAILED DEER presented by Laurie Ann Molitor has been accepted towards fulfillment of the requirements for M . S 0 degree in criminal Justice Majorp es r Date /Q7//f/I q? 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution , LIBRARY W“ Michigan State L University PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 11/00 c/CiRC/DataDquS-9J4 DEVELOPMENT OF A MICROSATELLITE MARKER PANEL FOR GENOTYPING MICHIGAN WHITE-TAILED DEER By Laurie Ann Molitor A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE School of Criminal Justice 1 999 impo 0f id: In for Chara poten [0 qu; docur reach. marks One 61c allow haiea Hi bffied ABSTRACT DEVELOPMENT OF A MICROSATELLITE MARKER PANEL FOR GENOTYPING MICHIGAN WHITE-TAILED DEER By Laurie Ann Molitor Deoxyribonucleic acid (DNA) microsatellite markers are becoming an increasingly important tool for uniquely identifying individuals. Forensic scientists face the challenge of identifying individuals to the exclusion of all others with a high degree of probability. In forensic wildlife cases, it is necessary to identify the animal involved in the crime by characterizing genetic variability among the species in order to obtain high exclusion potential. Wildlife animals are being poached and illegally imported at a rate too difficult to quantify because the suspect is rarely caught red-handed and there is very little documented data about the poaching problem. With the advent of polymerase chain reaction (PCR) and fluorescent detection methods, amplification of DNA microsatellite markers for identification purposes in forensic science is becoming a widely used method of genetic typing. The hypervariability of microsatellites with their widespread distribution and high abundance in the genome allows for genetically typing an individual. Three highly polymorphic loci were found in this study of Michigan white- tailed deer and were multiplexed together in one PCR assay and run simultaneously in one electrophoretic lane of an Applied Biosystems, Inc. (ABI) 377 DNA Sequencer, thus allowing for increased speed and low cost of analysis. The benefits of this study will have a positive impact on our ability to enforce wildlife laws, derive estimates of inbreeding within the population and assist in wildlife management strategies. Mic this and ACKNOWLEDGEMENTS I would like to thank Dr. Paul Coussens from the Department of Animal Science at Michigan State University for his guidance and assistance throughout the completion of this research project, and the Department of Natural Resources (DNR) and the US. Fish and Wildlife Service for funding this research project. I would like to thank the committee members: Dr. Jay Siegel, Dr. Frank Horvath, Dr. Christina DeJong and Dr. Charles Bama for their time and effort reviewing and providing suggestions concerning this research project. I would like to thank Dr. Kim Scribner and Dr. Robert Tempelman for their statistical expertise and Dr. Hsiao-Ching Liu and Dr. Christiane Hansen for sharing valuable knowledge and advice related to this research project. Finally, I would like to thank Dr. Hans Cheng from the USDA Avian Disease and Oncology Laboratory in East Lansing for the use of his laboratory equipment, necessary for the completion of this research project, and my friends for supporting and being patient with me during the completion of this master’s thesis. iii TABLE OF CONTENTS LIST OF TABLES .............................................................................. v LIST OF FIGURES ............................................................................ vi NONTECHNICAL SUMMARY ............................................................. 1 INTRODUCTION .............................................................................. 3 HISTORY OF “TYPING” TECHNOLOGY AND LITERATURE REVIEW. . . . .....6 METHODOLOGY ............................................................................. 43 FINDINGS ...................................................................................... 51 DISCUSSION ................................................................................ 107 FUTURE DIRECTIONS ..................................................................... 118 APPENDICES Appendix A - Agarose Gel Preparation Procedure ............................ 123 Appendix B — Tissue Extraction Procedure ..................................... 125 Appendix C - Acrylamide Gel Preparation Procedure ...................... 128 Appendix D — Multiplex PCR Protocol ........................................ 130 REFERENCES .............................................................................. 132 BIBLIOGRAPHY ........................................................................... 141 iv Tabb Tablt Tabl< Table Table Table Table Table Table Table Table Table Table Table Table Table LIST OF TABLES Table 1 Comparison of markers, techniques and methods of detection ................... 42 Table 2 Characteristics of the microsatellite markers used in this study ................. 45 Table 3 Amount of each component for a 25uL PCR ....................................... 46 Table 4 PCR thermocycler temperature programming ..................................... 47 Table 5 Initial screening results in base pairs from an ABI 37 7 of eight markers using random deer samples ........................................................... 59 Table 6 Allelic data for the nine pedigreed white-tailed deer families from Figure 14 ................................................................................ 66 Table 7 Michigan white-tailed deer database ................................................. 69 Table 8 Summary data for all 450 sampled Michigan white-tailed deer ................. 96 Table 9 - Statistical data testing Hardy-Weinberg equilibrium of Michigan white-tailed deer population subdivided into four regions of Michigan ....................... 97 Table 10 - Southern Michigan lower peninsula white-tailed deer data ............... ' ..... 100 Table 11 - Northern Michigan lower peninsula white-tailed deer data .................... 101 Table 12 - Michigan upper peninsula white-tailed deer data ............................... 102 Table 13 - North East Michigan lower peninsula white-tailed deer data .................. 103 Table 14 - North West Michigan lower peninsula white-tailed deer data ................ 104 Table 15 - Frequency of alleles for each marker in the four regions of Michigan ....... 105 Table 16 - F s, and Chi-Square values from comparing the frequency of alleles in the four regions of Michigan ........................................................... 106 Figuri HEUI: “Elite LIST OF FIGURES Figure 1 - Schematic diagram of polymerase chain reaction amplification ................ 20 Figure 2 - Schematic diagram representing the fluorescent intensity of alleles differing by one repeat unit of a dinucleotide microsatellite marker ....................... 30 Figure 3 - DNA quality of previously extracted genomic DNA samples .................. 52 Figure 4 - Additional genomic DNA samples previously extracted showing highly variable DNA quality ................................................................ 53 Figure 5 - Comparison of genomic DNA samples extracted using two different lysis buffer and enzymes .................................................................. 54 Figure 6 - Agarose gel of marker: JP15 ......................................................... 56 Figure 7 - Agarose gel of marker: CSN3 ....................................................... 56 Figure 8 - Agarose gel of marker: IRBP2 ...................................................... 57 Figure 9 - Agarose gel of markers: CRFA and FCB304 ..................................... 57 Figure 10 - Agarose gel of markers: IGFl and OBCAM ...................................... 58 Figure 11 - Agarose gel of marker: IP23 ......................................................... 58 Figure 12 - Heterozygosity of eight markers screened on twenty random deer samples..6l Figure 13 - Graphical representation of the degree of polymorphism for the three selected markers screened on twenty random deer samples .................... 62 Figure 14 - ABI gel file containing nine pedigreed white-tailed deer families ............. 65 Figure 15 — Example of an ABI gel file containing fifty samples with the three multiplexed markers ................................................................... 67 Figure 16 - Example of ABI electropherograms of five samples from Presque Isle ....... 68 Figure 17 - Heterozygosity of the three microsatellite markers for Michigan white-tailed Deer ..................................................................................... 90 Figure 18 - Graphical representation of IGF 1 alleles in the database ........................ 91 vi Ti, Fig LIST OF FIGURES (CONT’D) Figure 19 - Graphical representation of CRFA alleles in the database ...................... 92 Figure 20 - Graphical representation of OBCAM alleles in the database ................... 93 vii relia erid b‘pir [filler mate then CXClu dCVe} Wildli mdl\'i( Smite NONTECHNICAL SUMMARY Poaching, as defined by the Colorado Division of Wildlife, is the illegal taking or possession of any game, fish or non-game wildlife (Zumbo, 1999). Poaching entails the illegal hunting of wildlife on other people’s property, taking wildlife out of season or shooting more than the amount allowed. With any violation of poaching, the cost of these poaching crimes is well into the billions (Zumbo, 1999). Wildlife animals are being poached and illegally imported at a high rate and presently there is no economical, reliable, sensitive or time-efficient method of providing critical evidence linking evidentiary samples to an individual unless apprehended “red-handed”. The objective of this study is to develop a practical, economical, time-efficient DNA typing system for wildlife forensic scientists to utilize for individualizing forensic white- tailed deer evidence. In forensic wildlife cases it is most usual to “match” evidentiary material to material in the possession of the suspect (i.e. the gut pile at the crime scene to the meat in the suspect’s freezer or a drop of blood from the deer in the suspect’s possession). Forensic scientists face the challenge of identifying individuals to the exclusion of all others with a high degree of probability or with utmost certainty. To develop a DNA typing system that can positively place a suspect at the scene of a crime by matching two separate deer samples provides a powerful law enforcement tool for wildlife officials. Three regions of the deer’s DNA were found to be highly variable between individuals and in combination could distinguish an innocent suspect from a guilty suspect with an extremely high degree of probability. That is, there is almost 100 percent 1 dex' wil. enfc (pre crirr a 511: men ester is \‘ez Criier There after t certainty that the evidence at the crime scene can be matched to the evidence in the suspect’s possession. With the commission of any crime there needs to be a definitive method of placing the suspect at the scene of the crime and the DNA typing system developed in this project can do just that. This DNA typing system is a powerful tool for wildlife forensic scientists to utilize to match evidence samples and will assist law enforcement officers in enforcing wildlife laws and convicting poachers. The benefits of having this DNA typing system are twofold in that of deterrence (prevention before the act of the crime) and of enforcement (conviction after the act of the crime). The enforcement of wildlife laws can immediately benefit by being able to place a suspect at the scene of the crime. The effect of deterrence is not so obvious because monitoring the effect of something not happening is hard to do unless a solid database is established to monitor and evaluate the numbers. According to Jim Zumbo (1999) there is very little data about poaching and the data is not being collected using universal criteria amongst organizations or agencies. The data can thus not be evaluated accurately. Therefore, the goal of this DNA typing system is to assist in the conviction of poachers after they commit the crimes and to prevent or deter criminal activity from occurring in the future. A future goal would be to establish a universal database from which an accurate evaluation of the deterrent effect can be achieved. other the 0' anal} sunli; degra IO 1102 inclu: can b urn. SUSpe INTRODUCTION Forensic scientists face the challenge of identifying individuals to the exclusion of all others with a high degree of probability. Evidence found at a crime scene is not always in the optimal condition for many traditional scientific assays. Many samples collected for analysis have been exposed to environmental insults, such as extreme temperatures, sunlight or moisture, which degrade the biological components. Typically proteins degrade rapidly while DNA may be more stable for use in several techniques. Traditional serological techniques utilizing antigens, proteins or enzymes are limited to non-degraded samples and produce results with limited statistical probability of inclusion. Techniques using these biochemical markers are easy to perform and results can be scored unambiguously however, they are limited in their allelic variation which, in turn, lowers the statistical probabilities associated with matching the evidence to the suspect or victim. Traditional restriction fragment length polymorphism (RFLP) technique utilizing DNA is also limited to non-degraded samples. This technique, while still being used extensively, is limited to the use of high quality DNA. Evidence samples containing non- degraded DNA are analyzed by this technique, which result in a unique “individual- specific” DNA profile. The RFLP technique has one of the highest discriminatory potentials and probability of inclusion between evidence and suspect amongst all the molecular biology DNA testing technologies. PCR technology has superseded RFLP with its’ ability to analyze very small and even partially degraded DNA isolated from environmentally challenged evidence 3 ex'id mien dis-er obtair freeze geneu discrir fluoreg assistir Wache reliabic- Ci‘idem Oiimodc Crimm‘ n. typing S) tailed dl‘t’ samples. This technique is performed utilizing minute quantities of DNA recovered from evidentiary material, amplified in repetitive cycles of denaturation, hybridization with specific primer pairs and extension to create an exponential amount of target DNA. PCR technology amplifying microsatellite markers with fluorescent detection results in a highly powerful discriminatory tool for identification purposes. Combining several microsatellite markers in a single analysis has allowed this technology to approach the discriminatory potential and probability of RFLP technology. In forensic wildlife cases it is most usual to “match” evidentiary material to material obtained from the suspect (i.e. the gut pile at the crime scene to the meat in the suspect’s freezer). Matching evidentiary and suspect derived samples requires characterizing genetic variability among the species in order to find DNA regions that offer high discriminatory potential. The use of PCR amplifying microsatellite DNA markers and fluorescent detection is a strong combination for such a matching process and thus, assisting law enforcement officials in enforcing wildlife laws. Wildlife animals are being poached and illegally imported at a high rate and presently there is no economical, reliable, sensitive or time-efficient method of providing critical evidence linking evidentiary samples to an individual unless apprehended “red-handed”. The application of modern molecular biology techniques to DNA testing will help reduce the chance a criminal will evade conviction. The objective of this study is to develop a practical, economical, time-efficient DNA typing system for wildlife forensic scientists to utilize for individualizing forensic white- tailed deer evidence. This involves the development of a panel of highly polymorphic microsatellite markers among the Michigan white-tailed deer population. Microsatellite 4 (ca rep: 0m, amt pnn requi order proba eflnb thr ABI3‘ markers must show genetic diversity within the population in order to uniquely identify the individual and have high exclusion potential with regards to the evidence. The use of several highly polymorphic markers is necessary to achieve a high degree of probability of a match between the individual and the evidence. To arrive at this DNA typing system, eight microsatellite markers from bovine (cattle), ovine (sheep) and cervine (deer) origin were used to amplify DNA from a representative sampling of Michigan white-tailed deer to assess genetic diversity among the population. The sampling population consisted of five random deer samples from each of 83 counties and two islands in Michigan. The database thus contains 850 data points per marker providing a large database for probability estimates. These estimates require a determination of the frequency with which alleles occur in the population in order to individualize a sample and match it to evidentiary material with a high degree of probability. Of the eight markers tested, three were found to be highly polymorphic and exhibit high heterozygosity (i.e. > 70 %). In combination, these markers were able to be multiplexed together in one PCR and run simultaneously in one electrophoretic lane of an ABI 377 DNA Sequencer. the PM Sen anti E level grou; ques the A inher Levir disco beIWe Perfor reactic Each ( El acid p} Uansfe; agaTOSe 1955 TX 1m 0 HISTORY OF “TYPING” TECHNOLOGY AND LITERATURE REVIEW Semlomal Typing Techniques The beginning of human identification was established by using red blood cell antigen typing systems in order to facilitate disputed paternity cases at the biochemical level, with the following chronology listed in Melvin et al. (1988). The ABO blood group system discovered by Karl Landsteiner in 1901 was the start to resolving questioned paternity cases. Rubin Ottenberg in 1921 performed family studies applying the ABO typing system to paternity problems with the knowledge of Mendelian inheritance from von Dungern and Hirschfeld in 1911. Then in 1927, Landsteiner and Levine discovered another blood group system MN and in 1937 Landsteiner and Wiener discovered the Rh system. Several other red cell antigen systems were discovered between 1945 and 1965 given the names Kell, Kidd and Duffy. These systems are performed by immunological testing of allozymes using standard hemagglutination reactions of the antigen on the surface of the red blood cell to an antibody to the antigen. Each of these systems identifies between three and nine different phenotypes. Electrophoretic separations of red blood cell isoenzymes (e. g. phosphoglucomutase, acid phosphatase, adenylate kinase and adenosine deaminase) and serum proteins (e. g. transferrin, haptoglobin and properdin factor B) through a medium (cellulose acetate, agarose, starch or acrylamide) under a constant applied electric field were developed in 1955 (Melvin er a1., 1988). This technology has allowed for the separation of proteins based on their electrical charge properties, and are detected by an enzymatic reaction to the protein or by staining of the protein using colored dyes. lsoelectric focusing 6 3::es coding 1333110“ hi; regulation 1' X These hi and enzymes providing cm can be scorer satiation 1' Hi prtduets. \kh concerned \t' antigenicity ii? 110‘. (listing. electrophoresis using a continual and linear pH gradient was developed in the 1970’s in order to separate proteins with increased resolution and decreased electrophoretic problems. Also developed in the 1970’s was the human leukocyte antigen (HLA) system showing a higher degree of polymorphism compared to previously mentioned systems. Genes coding for the HLA antigens reside at four loci and thus, exhibit high variability and show high exclusion potential due to the low frequency of allelic variants in the population (Melvin et al., 1988). These biochemical methods of differentiating individuals using antigens, proteins and enzymes are very powerful tools in identifying differences between individuals and providing exclusionary information. These techniques are easy to perform and the results can be scored unambiguously. However, they are systems limited in both their allelic variation (which limits the statistical probability of inclusion) and their use of protein products, which invariably degrade over time. Antigen-antibody systems have to be concerned withthe intensity of the reaction due to loss of antigen in the sample or weak antigenicity. Proteins and enzymes are not stable molecules and thus are denatured easily therefore null results can be misleading from the testing of samples using these biological components. Contamination from mixed samples and rare cross reactivity between different marker systems may also produce erroneous results. Immunologic assays can not distinguish between all variations in amino acids and are not as sensitive as DNA testing assays. DNA is highly stable, can be isolated from both living and dead cells and codes for the synthesis of these secondary by-products using an exact blueprint for all the biological components necessary for building and maintaining the life of the organism. In addition, every nucleated cell in the body contains the same genetic “blueprint”. 7 Tr of ha i_] at L Kra o o o o ' l ' I ' l h ° h se Tbhjg dchVe Traditional serologic testing has been revolutionized by the use of DNA testing. The use of DNA is by far more preferred because the code itself is being tested and the results have greater discriminatory power. A Brief History of DNA Advancements While advances at the protein level were continuing to expand, so were the advances at the DNA (deoxyribonucleic acid) level according to the following chronology listed in Krawczak and Schmidtke (1994). o In 1944 Avery and co-workers discovered that DNA is the genetic material, the “blueprint of life” and is in every nucleated cell of all living organisms. o In 1953 Watson and Crick proposed a model for the structure of DNA. 0 In 1961 Nirenberg and Matthaei deciphered the genetic code. 0 In 1970 Arber, Nathans and Smith discovered restriction enzymes which cut DNA at specific sites. 0 In 1972 Berg and co-workers developed molecular cloning of DNA. 0 In 1977 Sanger and co-workers developed methods for sequencing DNA. 0 In 1979 EM. Southern developed a method of transferring single-stranded DNA fragments to a more permanent membrane called Southern blotting. o In 1985 Kary Mullis and co-workers at Cetus discovered the amplification of DNA segments by PCR. This is only a partial list of the contributions to science since the beginning of DNA discovery in 1944. The applications of these key contributions to the field of molecular biology will be discussed in this chapter, but for now a discussion of DNA is necessary. C}'I( neo prot' grOL thyn and 2 mole Slllgl: nucle baekb and ar Purine [his is mainra but 011: base pa OfHUcl lumber DNA Structure DNA is a long polymer comprised of four subunits, adenine (A), guanine (G), cytosine (C) and thymine (T) found in all nucleated cells. DNA encodes all information necessary for the primary structure of proteins and thus helps direct the necessary life processes of an organism. DNA subunits called nucleotides are classified into two groups, the purines are adenine and guanine and the pyrimidines are cytosine and thymine, with each nucleotide containing a phosphoric acid group, a deoxyribose sugar and a nitrogenous base. Both purines and pyrimidines are heterocyclic, flat, planar molecules with the purines having a double ring structure and the pyrimidines having a single ring structure which allows the bases to stack one on top of each other. Each nucleotide is joined together by their sugar and phosphate groups forming a repeating backbone of sugar-phosphate-sugar-phosphate, etc. along each strand of DNA. Two strands make up the double stranded DNA molecule which are antiparallel to each other and are hydrogen bonded together by adjacent nucleotides on opposite strands. The purine (A) on one strand will always bind to the pyrimidine (T) on the other strand and this is called a base-pair, as with (C) to (G) according to Chargaff’s rule in order to maintain a stable DNA molecule. Therefore, each strand is complementary to the other but oriented in opposite directions along the duplex helical molecule. This specificity of base pairing allows for the storage and transfer of genetic information based on the order of nucleotides in each strand of DNA. The DNA sequence specifies the exact genetic instructions required to create a totally unique organism. 1C3 DNA Classification DNA can be put into three classes based on the function and characteristics of the nucleotide sequence. The first and smallest class, comprising only about 3% of the total genome, contains the unique sequences that are rarely repeated, representing the coding regions for genes (Frank, 1997). These sequences carry the genetic information necessary for the synthesis of proteins required for building and maintaining the metabolic processes in an organism. The next class contains sequences, which are moderately repeated at least 1,000 times, are located adjacent to unique DNA and are dispersed throughout the genome. These gene-related sequences for the RNA components of ribosomes, such as tRN A and rRNA, along with histones are considered noncoding DNA (Lindquester, 1997). The last class contains highly repetitive sequences, which are repeated thousands to even millions of times and are found in certain regions of the genome. Repetitive DNA This repetitive or noncoding DNA, often referred to as ‘junk’ DNA because it serves no function in protein synthesis, can be further classified into groups based on whether the repeat sequences are situated in tandem or interspersed within the genome. Interspersed elements are single units dispersed throughout the genome, usually not in tandem, but occur hundreds to thousands of times within the untranslated intronic regions of the genome. The most abundant sequence in the human genome associated with GC rich regions is called an Alu repeat element because the sequence contains the enzyme Alu I restriction site (AGCT) and is considered a short interspersed nuclear element (SINE), less than 500 bp long (Kobilinsky, 1993). The L1 or Kpn repeat element is 10 associated with AT rich regions because the sequence contains the enzyme Kpn I restriction site (GGTACC) and is considered a long interspersed nuclear element (LINE), greater than 500 bp long (Kobilinsky, 1993). Tandem Repetitive DNA Tandemly repeated or clustered sequences contain core sequences repeated numerous times and arranged side by side thus, in tandem. These core sequences, usually between two and six bases long (Tautz, 1989), repeated between ten and a hundred times, scattered throughout the genome millions of times, but localized to the centromeric region of the heterochromatin of chromosomes are called microsatellites or short tandem repeats (STR’s). Minisatellites, often associated with the term VNTR, have core sequences ranging from 10 to 50 bases long, repeated two to several hundred times and are mainly clustered in the proterrninal or telomeric regions of chromosomes, but have also been found in interstitial regions (Royle et al., 1988). Minisatellite Description Minisatellite sequences can vary in both the sequence of nucleotides in the repeat and the number of repeat units and thus, with variable numbers of tandem repeats are called VNTR’s. These hypervariable regions are excellent genetic markers due to their high polymorphic nature and their discriminating power for characterizing DNA. Minisatellites are thought to arise from either unequal exchange during mitosis between sister chromatids or during meiosis between homologous chromosomes (Jeffreys et a1. , 1985a). These minisatellite sequences may be responsible for promoting recombination events similar to the chi sequence (GGGCAGGAXG) in Escherichia coli (Jeffreys et aI. , 1985a). The GC rich core sequence of minisatellites are considered to be a recombination ll ll hot 56) res; p01: enzy six 0 or eli Si: globin Single 1 Z} gosh) results. 1 TWO d1 0‘. 11C gene in Mme, 1 ”Were: D\A ‘f‘, hotspot in human DNA (Jeffreys et al., 1985a). VNTR’s can be located at one locus or at several loci thus, given the names single-locus VNTR’s and multi-locus VNTR’s. respectively, and these genetic markers are identified by restriction fragment length polymorphisms (RFLP). RFLP Description RF LP’s are variations in the length of DNA fragments as a result of a restriction enzyme cleaving the genomic DNA at a certain sequence recognition site, usually four, six or eight bases long. Variation is either due to a single nucleotide substitution creating or eliminating a cleavage site for a specific restriction endonuclease or a rearrangement of a DNA segment accounting for the variable length fragment. RFLP’s are inherited dominantly in a Mendelian fashion, one allele from each parent, and are only dimorphic, that is, showing the presence or absence of a restriction site detected by length polymorphisms. Single locus VNTR’s were first analyzed by using RFLP’s adjacent to the beta- globin locus in human DNA associated with sickle cell anemia (Kan and Dozy, 1978). Single locus VNTR’s create only one or two bands depending on the individual’s zygosity, which allows for unambiguous interpretation of the results. Single locus VNTR results, however, are not very informative because each individual will have only one or two different bands. The first highly polymorphic locus was identified in a nonspecific gene in human DNA showing high variability with at least eight bands (Wyman and White, 1980). Using a multi-locus VNTR, present at multiple loci in the genome, scattered throughout the telomeric ends of chromosomes, creates an individual-specific DNA ‘fingerprint’ resulting in multiple bands which are unique to an individual (Jeffreys 12 ell anz V5 ma; whc' locr M in ar idem begir gene: et al., 1985a). The work done by Alec Jeffreys initiated interest by forensic scientists to analyze forensic specimens using RF LP DNA typing. The use of several single-locus VNTR’s (Nakamura et al., 1987) simultaneously will also create a unique DNA ‘fingerprint’ for identification purposes however, interpretation of the resulting pattern may be problematic due to possible band sharing between loci. Band sharing occurs when the size of bands from one locus overlap at the same size as bands from another locus, thus, creating ambiguous interpretation of the results. RFLP Procedure The RFLP DNA typing technique is an extensive multi-step procedure which results in an autoradiograph or fluorograph containing a unique DNA fragment pattern (except in identical twins) when several DNA probes are used or a multi-locus probe is used. The beginning of this procedure requires high quality and quantity, at least 5-10 ug of genomic DNA. The isolation and purification of the DNA can be performed using standard organic procedures (Sambrook et al., 1989) or by using commercially available nonhazardous kits resulting in high molecular weight DNA, relatively free of proteins and not sheared into small pieces. The DNA is then digested with restriction enzyme(s) (RE) selected based on the frequency with which they cut the DNA and the sequence of their recognition site, so as to not cut within the probing sequence. The RE digested DNA is electrophoresed overnight, applying low voltage, through a low percentage agarose gel in order to separate the fragments based on size. The double- stranded DNA fragments in the gel are then denatured to become single-stranded by soaking the gel in an alkaline denaturing solution. It is necessary to transfer single- 13 strand hybric knom transfc ultrav the m: single probe compl condit L; the siz bands detem immig cell an. I Andr stranded fragments to a sturdier membrane, usually nylon or nitrocellulose, so that hybridization of the single-stranded probe can be achieved. This transfer process is known as Southern blotting (Southern, 1975) and requires capillary action to perform the transfer. The membrane is then baked in an oven (nitrocellulose) or exposed to ultraviolet light (nylon) in order to cross-link or permanently fix the DNA fragments onto the membrane. Hybridization of the membrane-bound denatured DNA fragments to the single-stranded complementary sequence of the radioactive or chemiluminescent labeled probe is performed under optimal conditions for specific binding of the probe to the complementary sequence. Unbound probe is washed away under varying stringency conditions. Lastly, the membrane bound with the labeled probe is exposed to x-ray film to detect the size of fragments showing complementarity to the probe. The resulting pattern of bands created by this RF LP DNA typing technique has proved useful for comparison in determination of paternity (Jeffreys et al., 1985b; Wells et al., 1989), in solving immigration cases (Jeffreys et al., 1985c), in diagnosing medical diseases such as sickle cell anemia (Chang and Kan, 1982) and directly assisting in the first criminal case (State v Andrews, 1987) resulting in a conviction based on VNTR DNA testing evidence. VNTR was the first DNA evidence technique accepted by most courts, including those in Michigan. DNA Figerprinting in Forensic Casework Forensic casework has gone through a major transition from traditional serology to RF LP DNA testing since the work done by Alec Jeffreys and coworkers in 1985 describing ‘DNA fingerprinting’ and its potential use in forensic science. Lifecodes l4 C 0 cas the the reel Stat area the s ques guid. case guide coun the sc the Li fOr f0: assma DNA, lab0rat labofatt Company in 1986 and Cellmark Diagnostics in 1987 both took interest in accepting casework by performing RFLP technology in their laboratories (Weedn, 1993). In 1988 the Federal Bureau of Investigation (FBI) started using RFLP for casework, along with the first state crime laboratory in Quantico, Virginia in 1989 (Weedn, 1993). This new technology has sparked interest from crime laboratories in every state in the United States, along with other private companies investing into this technologically advanced area. Guidelines for DNA Testing With any new technology, the reviews and criticisms are always there to follow from the scientific community, criminal justice professionals and the average intelligent person questioning the reliability and accuracy of this technique and any other DNA testing. The guidelines for the admissibility of scientific evidence were established in 1923 with the case United States v. Frye, also called the “Frye” hearings (Baird, 1992). These guidelines are used in determining whether DNA evidence should be admissible in a court of law based on the premise that there is general acceptance of the evidence from the scientific community. In 1990, the Office of Technology Assessment which is part of the United States Congress concluded that DNA evidence is reliable and can be utilized for forensic casework only if appropriate technology, quality control and quality assurance procedures are implemented (Weedn, 1993). The Technical Working Group on DNA Analysis Methods (TWGDAM) represented by scientists from North American laboratories developed DNA methodology and quality assurance guidelines for laboratories to follow for forensic RF LP typing (TWGDAM, 1989). Included within these guidelines is the application of external proficiency testing by a reputable laboratory 15 0TH and ino Resi TWt Cour influ Simi have andP These or accrediting agency (TWGDAM, 1990), such as the College of American Pathologists and the American Society of Crime Laboratory Directors Laboratory Accreditation Board in order to assure quality performance of the laboratory (Weedn, 1993). The National Research Council in 1992 issued a report stressing the guidelines developed by the TWGDAM regarding standardized laboratory procedures in 1990 (National Research Council, 1992). The American Society of Crime Laboratory Directors has also been influential in making recommendations regarding DNA technology in forensic science. Similar guidelines, quality assurance and accreditation programs for PCR technology have also been established by the TWGDAM. Revisions to these guidelines for RFLP and PCR DNA testing will be necessary as the technologies and experience advances. These methodologies are now widely accepted in the criminal justice community when appropriate quality control methods are followed however, current controversy focuses on potential human and technical errors and on the statistical interpretation of results (National Research Council, 1996; Weir, 1996). PCR Description The amplification of specific DNA segments by PCR discovered in 1985 by Kary Mullis and co-workers at Cetus has revolutionized molecular biology and is rapidly replacing RFLP DNA typing for identification of forensic evidence. This in vitro enzymatic amplification of a DNA segment is performed utilizing minute quantities of DNA in repetitive cycles of denaturation, hybridization and extension creating an exponential amount of target DNA. This amplified DNA can then be directly sequenced to detect single base polymorphisms, electrophoresed to detect sequence length variations or hybridized to allele-specific probes to detect sequence polymorphisms. Forensic l6 finer quah IX§A LD§A 10“"r avafl. nneq genor genes regnr secfic nanog micro thx heme. (“hyh scientists have benefited tremendously by this new technology because the quantity and quality of recoverable DNA found at crime scenes is often less than-optimal. PCR technology does not require high molecular weight DNA or large quantities of DNA like RF LP technology. PCR can be performed with only a few copies of a target DNA sequence as long as the target sequence to be amplified is not degraded or exists as low molecular weight DNA. Degradation of DNA for RFLP analysis limits the availability of restriction sites for the enzyme being used which in turn affects the interpretation of the results. PCR can be performed with degraded DNA because 1 pg of genomic mammalian DNA corresponds to approximately 3 x 105 copies of autosomal genes (Cha and Thilly, 1993). The chance that each copy shows degradation in the same region to be amplified is very small. Thus, in any sample, there is likely to be several sections that are suitable for amplification. Most PCR applications only require nanogram quantities of DNA per reaction as compared to RF LP requiring several micrograms. PCR amplification is sensitive to interfering polymerase inhibitors found in DNA samples extracted from materials containing forensic evidence such as detergents, heme, melanin pigments, dyestuffs, sodium acetate, metal cations, urea or EDTA (ethylenedinitrilo tetraacetic acid) (Sensabaugh and Blake, 1993). The sensitivity of PCR to contamination is of major concern because only minute amounts of extraneous human DNA may cause erroneous results. Contamination often comes from mixed samples at the crime scene, which is a reality for forensic scientists to deal with. Another source is from laboratory personnel or crime scene technicians and investigators introducing their own DNA into the evidence. This can be a problem if the 17 et’ide coma pnor requir proce conta PC‘R T the sp the su‘ Other PCR( arnplif [hemlt‘ evidence sample contains a very small amount of DNA. The last major source of contamination arises when other amplified samples are accidentally mixed with evidence prior to amplification. This can be a serious problem because only an aerosol droplet is required to cause preferential amplification of the contaminant. Careful laboratory procedures need to be adopted to minimize these sources of contamination. Many contamination errors are detectable and can be corrected by reanalyzing the evidence. PCR Procedure The PCR procedure, first developed as a technique by Saiki et al. in 1985 to amplify the specific beta-globin sequence, is a cyclic process that does nothing more than increase the subanalytical quantities of DNA to a level that can be detected by routine methods. Other studies quickly followed using enzymatic amplification of DNA in vitro by the PCR (Mullis et al., 1986; Saiki et al., 1986; Mullis and Faloona, 1987). The amplification procedure is a relatively simple laboratory technique to perform with a thermocycler doing most the work. The thermocycler is a programmable machine, which has the ability to cycle through various temperatures (0-100° C) in a relatively short amount of time with temperature homogeneity and accuracy. The sample preparation includes double-stranded DNA to be amplified, two single-stranded oligonucleotide primers flanking this DNA segment, a DNA polymerase, deoxynucleotide triphosphates (dNTP’s), a buffer, magnesium chloride (MgClz), salts and deionized water. The three step cyclic procedure begins with the denaturation step to separate the double-stranded DNA molecules into single strands by heating them to 94-95° C for one to three minutes. This creates two strands of DNA, which are both used as templates for the synthesis of complementary strands of DNA. The temperature is then lowered to 18 temp temp doub nucle optim range T Order 1 Cycle. and aft moleci Figure becaus. [he lei); PTOdUC Mime produc both p. allow the oligonucleotide primers (stretches of up to twenty nucleotides in length), to find and hybridize to their complementary sequences on opposite strands flanking the sequence to be amplified, called the annealing step. The last step involves the extension of the primers at the 3’ end by the addition of nucleotides complementary to the target sequence mediated by a DNA polymerase, thus called the extension step. The temperature of the annealing step will vary between 45-65° C depending on the melting temperature T m (the temperature at which half of the primer is single-stranded and half is double-stranded) of the oligonucleotide primers, which is directly related to their nucleotide length and content. The temperature of the extension step is dependent on the optimal temperature for the activity of the DNA polymerase being used, usually in the range of 70-75° C. This three-step amplification process is cycled several times, usually 25 to 30 in order to generate millions of copies of the target sequence. After the first round of a cycle, one double-stranded molecule has been doubled to two double-stranded molecules and after the second round of a cycle, the two molecules have been doubled to four molecules and so on, an exponential (2") fold increase, for each additional cycle (see Figure 1). The number of target sequence has in effect been doubled after each cycle because each strand serves as a template for replication in subsequent cycles. However, the length of the products generated after the first cycle are longer than the desired product size because the polymerase extends until the denaturation step forces the polymerase to separate from the DNA template. In subsequent cycles the short or desired product size increases exponentially because the ends of the product have been defined by both primers, whereas the original double-stranded molecule only increases linearly. l9 3’ 5’ double-stranded 5/ , 3/ target DNA cycle 1 i strand denaturation single 3/ 5/ 5/ 3/ stranded * DNA i primer annealing and extension Primer 1 Primer 2 double — > < _ stranded 3! ' 5/ 5/ 3/ DNA (2 copies) cycle 2 i strand denaturation 5/- % f _ 5/ single 3’ 3’ stranded 3’ SI 5/ 3/ DNA l primer annealing and extension Primer 2 Primer 1 { _ — > - 4 ¢ — double 5’ 5’ 3/ 3/ stranded Primer 1 Primer 2 DNA — , ‘ — (4 copies) 3’ 5’ SI 3’ i after 2 cycles of amplification I 31— _ 3 3i: _ 3] double-stranded DNA with defined ends including primer sequences (2 copies) and 3/ 3’ 3” y double-stranded DNA with one end defined (2 copies) Figure 1. Schematic diagram of polymerase chain reaction (PCR) amplification. 20 minute optimi amplif thermc the am every 1 of Mg( amplif indit'id H mm th ICmPer initial t Contem Primer “36d. ft Offimt ampljf: “lens; PCR, with its specificity to the desired target sequence and extreme sensitivity to minute quantities of target DNA, is a technique that requires many components to be optimized in order to obtain the desired product and only the desired product. The amplification conditions, containing both the components in the reaction and the thermocycling temperatures, times and number of cycles, are variables that directly affect the amplification process. These variables need to be optimized for almost every locus studied, unless similarity exists between marker type, primer T m and amount of MgCl2 required for each reaction. Therefore, much time is spent optimizing the amplification conditions prior to validating a marker system for use in population studies, individual identification or other PCR applications. PCR Thermocycler Programming The temperature and amount of time selected for the thermocycling conditions, along with the number of cycles are dependent on the following criteria. The denaturation temperature should be close to boiling in order to completely separate the strands with an initial time of a few minutes. The annealing temperature depends on the length and GC content of the primers and, in general, should be around five degrees below the Tm of the primer pair. The extension temperature should be selected based on the polymerase being used, for Thermus aquaticus ( T aq) the optimal temperature would be 72° C. The amount of time for this step is based on the length of the target DNA being amplified. That is, amplifying a fragment greater than le (kilobase) in length would require more extension time for the polymerase to complete the full sequence, otherwise a standard time for smaller fragments would be 30 seconds to 1 minute for each step. The number 21 of cycles depends on the amount of product generated based on the amount of starting template, which can vary from as little as 20 cycles to as many as 35 or even 40 cycles. PCR Components The components of a standard PCR are contained in a reaction volume of 25, 50 or 100 pl with 50 to 100 ng of genomic DNA mixed with 50 mM KCl, 10 mM Tris HCl pH 8.4, 0.25 uM of each primer, 200 uM of each dNTP (deoxynucleoside triphosphate), 1.5 mM MgC12, 2.5 units of T aq polymerase, deionized water and overlayed with a few drops of mineral oil (Saiki, 1989). The mineral oil is added to reactions when the thermocycler being used does not have the heated lid feature to prevent evaporation of the reaction mixture and to increase the rate of thermal equilibration. These are standard conditions that may not provide adequate results in all situations. Modifications may be necessary to obtain the desired specificity and yield of PCR product. The component with the most dramatic effect on specificity and yield is the amount of MgCl2 in the reaction, that is, the amount of free Mg++ available for Taq polymerase. The effect of dNTP’s chelating magnesium will lower the optimal concentration of magnesium. If all the Mg++ is bound by the dNTP’s, then none will be present to facilitate T aq in amplifying the target DNA. The absence of a PCR product can often be corrected by adding a higher concentration of MgCl2 to the reaction, while the presence of too many products is the result of too high of MgClz. Optimal levels of free Mg++ should be around 1.0 mM for most PCR’s. Concentrations of Mg++ approaching 10 mM MgCl2 inhibit the activity of T aq (Gelfand, 1989). 22 dNTP arnpli Mg+c 1 Kb. inhibi these Gorc Because they act as building blocks for the amplification product, the amount of dNTP’s should always be in excess, 200 uM, of DNA target sequences for efficient amplification but, it is not recommended to increase this amount because it will chelate Mg++- as mentioned above. However, if amplifying a large target sequence, greater than 1 Kb, 3 higher concentration of dNTP’s is necessary. T aq polymerase is actually inhibited in the presence of millimolar concentrations of dNTP’s (Gelfand, 1989) and these high dNTP concentrations may actually promote misincorporations. The selection of oligonucleotide primers is of paramount importance to the success of an amplification reaction. The criteria for primer design entails creating both primers which are between 20 and 30 bases in length with a GC content between 40 and 50 percent and with a random base distribution not containing stretches of similar bases. A 20mer has a specificity of 1/420 in locating the exact complementary sequence in the template DNA and with a 50 percent GC content has a Tm range of 56-62° C (Cha and Thilly, 1993). Each additional nucleotide will increase the specificity of the primer by a factor of four and increase the TI“ by two or four degrees depending on which base is added. Primers should not be able to form secondary structures within themselves because then they will not be able to hybridize to the template DNA. They also should not have complementarity to each other in order to avoid the formation of primer dimers. This is of major concern when using multiple primer pairs in one reaction (multiplex). Perfect complementarity should exist at the 3’ end of the primer, usually the presence of a G or C at the very end will assure primer extension by the polymerase because these bases form stronger bonds and will anchor the primer .to the template. Lastly, each 23 pri bet D.‘ at! aft for adi €111 des HOT Pele 01h£ 'Dro primer should be in a ten-fold excess of the target sequence during PCR (Cha and Thilly, 1993). The specificity, sensitivity. yield and length of PCR products being amplified has been substantially improved with the replacement of the Klenow fragment of E. coli DNA polymerase I with an enzyme isolated from the thermophilic bacterium T hermus aquaticus (Taq) (Saiki et al., 1988). Prior to this discovery, fresh Klenow enzyme was added to the reaction mixture after each cycle of denaturation because this enzyme is heat-sensitive and would have no activity after one round of amplification. This sequential addition of polymerase to the reaction mixture has been eliminated with the discovery of T aq, which is very thermostable with repeated exposure to temperatures of 95° C. This allows the PCR cycling to be fully automated using thermal cycling machines and increases the yield of product because the enzyme still maintains activity after numerous cycles. The processivity of T aq polymerase during DNA synthesis allows for the amplification of longer DNA segments, up to 10 Kb (Jeffreys et al., 1988), which adds to the list of PCR applications. The specificity and sensitivity has been greatly enhanced by the use of Taq polymerase because at higher temperatures, the annealing of complementary primers to the template DNA will result in only amplification of the desired target sequence with an increase in the yield of that product, eliminating nonspecific amplification. The fidelity of T aq polymerase in PCR amplification is not a strong feature with a relatively high error rate of 10'5 nucleotides/cycle (Gelfand and White, 1990) compared to other polymerases. This misincorporation of nucleotides is due to the lack of the ‘proofreading’ 3’ to 5’ exonuclease activity of Taq polymerase. Misincorporation of a 24 nucle temp PC R errors becat detec fewer probl: mutat pol yn nucle. nucleotide would have to be during the early stages of PCR, with very limited amount of template, affecting many copies, in order to have a significant effect on the amount of PCR products detectable by electrophoretically visible bands (Saiki et al., 1988). The errors made by Taq polymerase should not be a problem for many PCR applications because the analysis of several samples to establish a consensus sequence is performed to detect any misincorporated nucleotides. Using more initial template DNA molecules, fewer cycles of amplification and modifying the reaction conditions can reduce this problem exhibited by T aq polymerase. Other applications, such as cloning or point mutation analysis where fidelity is of concern, may require the use of the Pfiz DNA polymerase isolated from Pyrococcusfirriosus with an error rate of 6.5 x 10'7 nucleotide/cycle (Andre etal., 1997). However, the therrnostability, processivity and specificity of T aq polymerase has made it the most utilized polymerase in PCR to generate large quantities of a specific target sequence. Sources of Error During Amplification of Microsatellites PCR amplification of dinucleotide microsatellite markers using T aq polymerase presents two sources or error in genotyping studies. The catalysis of a nontemplated addition of a nucleotide, predominantly adenosine, to the 3’ end of the amplified PCR product (Clark, 1988) and the appearance of shadow bands with the PCR products separated by intervals of two nucleotides (Hauge and Litt, 1993; Murray et al., 1993). Both of these can cause problems when genotyping the sizes of dinucleotide microsatellite fragments generated by PCR. However, strategies have been proposed to deal with these biological occurrences. 25 Tl differ: additic our? know al., 19 source signifi produc Which famrl an add Modifl facilit; are all exOnt may repeat anal}, Perm. Slipp: The addition of the nontemplated nucleotide to the PCR product creates bands which differ in size by a single base and is primer specific (Smith et al., 1995). The degree of addition varies from marker to marker with some markers not affected at all and some only partially affected with both product sizes being present in similar quantities. The known fact that this situation occurs with some markers, PCR based strategies (Smith et al., 1995) and primer modifications (Brownstein et al., 1996) strategies to minimize this source of error have been addressed. Changing the PCR thermal cycling conditions can significantly reduce the error rate of inconsistent allele calling. To minimize the production of allele + A (adenine) products, a two step cycling protocol can be used which eliminates the extension step that causes the polymerase to add the extra A. To favor production of the allele + A product, a three step cycling protocol can be used with an additional final extension step facilitating the nontemplated nucleotide addition. Modifying the reverse primer on the 5’ end by adding the sequence GTTTCTT can facilitate adenylation of the 3’ end of the forward primer, thus generating products which are all allele + A (Brownstein et al., 1996). Other methods employing an additional enzymatic step after amplification by adding T4 DNA polymerase with a 3’ to 5’ exonuclease activity to remove the unpaired base have been proposed (Kimpton er al., 1993). The characteristic feature of T aq polymerase is to frequently skip or occasionally add repeat units during the extension step of PCR amplification causing great difficulty in analyzing dinucleotide microsatellite markers. Other short tandem repeats (tri-, tetra- or pentanucleotides) exhibit this skipping by T aq polymerase very infrequently. This slipped strand mispairing by T aq is the major mechanism for the generation of shadow 26 bands. (Hang: itselff shorter e! 01. if band a the act amplif unit. I thermc lOCi Q Seque Team; The p 0\‘er' It JOCus bands, which are less intense and intervals of two nucleotides short of the correct allele (Hauge and Litt, 1993). This slippage by Taq results from the temporary dissociation of itself from the template strand allowing repeats in the template to form a loop, which shortens the PCR product by the number of repeat units in the loop. The study by Murray et al. in 1993 also supports this mechanism because they sequenced the actual shadow band and found it to contain the flanking sequences on both sides of the repeat, but that the actual repeat sequence was ambiguous. This suggests that the shadow band is the amplified product with the repeat sequence being scrambled, but still short by one repeat unit. Presently, the only correction to the occurrence of these shadow bands is to use a thermostable DNA polymerase with a higher processivity than T aq. Multiplex PCR The ability of PCR to be extended even further to the simultaneous amplification of multiple loci using the same DNA template in a single PCR reaction, called multiplex PCR, has greatly increased the throughput of data in considerably less time (Chamberlain et al., 1988). However, more effort is required to develop optimal conditions for the combination of primer pairs being amplified simultaneously. Multiplexing two or more loci often can be performed under the same reaction conditions used to amplify the sequences separately. The main consideration is that the components of the amplification reactions and the temperature cycling conditions are the same for all the loci involved. The primers must not show homology to each other and the product sizes should not overlap each other. All the factors discussed previously for a single amplification of one locus must also be considered when optimizing a multiplex PCR. The adjustment of 27 these avail; Mult S}'SI€1 1, Kim 1 Walla micro With E many semi-2 fluoro. rCSpec arnplif of a D Specifi length these variables is specific to the unique loci being amplified and general guidelines are available to assist the scientist in optimizing the multiplex PCR (Henegariu et al., 1997). Multiplex PCR and Fluorescent Detection The combination of multiplex PCR and semi-automated fluorescent detection systems has become a rapid and powerful technique for individual identification (Kimpton et al., 1993). The amplification of loci consisting of two alleles (Skolnick and Wallace, 1988) has been extended to the amplification of highly polymorphic microsatellite loci and genotyping these loci using automated DNA sizing technology with multi-color fluorescent detection (Ziegle et al., 1992). This ability to coamplify many loci in one PCR has been facilitated by the use of fluorescently labeled primers and semi-automated fluorescent DNA sequencers. Oligonucleotide primers are labeled at their 5’ end with one of three possible fluorochrome dye molecules: Fam, Hex or Tet, fluorescing blue, yellow and green, respectively. The labeling of the primers at the 5’ end does not effect the PCR amplification, but it does allow for the simultaneous analysis of multiple loci in one lane of a DNA sequencing gel. The fluorescent dye is only on one primer of each locus- specific primer pair, since both primers are amplifying the same target sequence the length will be the same. The use of an internal size standard, labeled with the red fluorescent dye TAMRA, in every gel lane is referenced when calling the fragment sizes. The benefit of this is to eliminate interpretation errors due to electrophoretic mobility differences between lanes across the gel and thus, increasing the size calling accuracy of the alleles. Fragments present in each lane are automatically sized against the internal size standard comigrating 28 inea TAX convl er a]. advar have abilit; quant intens er 01. lane. Carine ofPC for a 9316: of th; in each lane, which compensates for any gel distortions in that lane. Size standard TAMRA 350 from ABI, for example, contains known fragments with sizes: 50, 75, 100, 139, 150, 160, 200, 250, 300,- 340 and 350, representing fragment lengths in nucleotide base pairs. Fluorescence-based detection of microsatellite PCR products compared with conventional autoradiographic methods (Schwengel et al., 1994) and silver staining (Lins et al., 1996; Budowle et al., 1997) for identification of individuals has resulted in many advantages. Fluorescent compounds are safer to use, easier and cheaper to dispose of and have a longer shelf life than radioisotopes. They are extremely more sensitive with the ability to detect picomole quantities of fluorescently labeled primers and picogram quantities of initial template DNA. Fluorescent signals are linear over a wider range of intensities, where signal strengths of multiple loci greatly vary in magnitude (Schwengel et al., 1994). Multiple loci can be simultaneously analyzed in a single electrophoretic lane, whereas with radioisotopes and silver staining this variation in signal intensity cannot be tolerated. The signal intensity of the peaks can be used to estimate the amount of PCR product present. Scoring alleles of a heterozygous individual being genotyped for a dinucleotide microsatellite marker with alleles differing by one repeat unit is much easier with fluorescence. The intensity of the smaller allele overlapping the stutter band of the larger allele will be greater than the intensity of the larger allele (see Figure 2). 29 5} tr. si. [11 U1 a“.- [‘1 dis flu 3p; Allele Size true allele - 220 bp - true allele stutter band 218 b and true allele - P — stutter band stutter band — 216 bp Heterozygote Homozygote Figure 2. Schematic diagram representing the fluorescent intensity of alleles differing by one repeat unit of a dinucleotide microsatellite marker. The time required to score alleles and the number of genotyping errors drop considerably due to the combination of automated fluorescence-based electrophoretic systems and genotyping analysis software. Genotyping analysis is being facilitated by the use of internal size standards and analysis software for the accurate assignment of fragment sizes and the unambiguous scoring of alleles. Ghosh et al. in 1997 describe methods for accurate sizing of alleles in order to reduce genotyping error rates. Manual methods of scoring autoradiographs or gels stained with silver lead to more typing and sizing errors because both DNA strands are being detected. These methods are extremely time-consuming and are not compatible with high throughput genotyping. Several validation studies, for use in forensic applications, have been performed utilizing multiplex PCR of short tandem repeats with fluorescent detection (F regeau et al., 1999; Budowle et al., 1997). These studies support the use and benefit of this genetic typing system for forensic identification and according to Fregeau et al., could provide discriminatory power of approximately 0.9999. This discriminatory potential of fluorescent multiplex STR marker systems in human identification is the basis for applying this technology to individualize evidence samples in forensic wildlife cases. 30 01 8! CC OI I: oli tra H} the C03 Multiplex PCR using microsatellite markers for characterizing genetic variability among Michigan white-tailed deer, for the purpose of individualizing evidence samples, should be both cost-effective and beneficial. The homology between microsatellite loci is often conserved among related species (Moore et al., 1991), saving time and effort in primer development. Bovine microsatellites are highly conserved among Red deer (Kuhn et al., 1996) and Sika deer (Slate et al., 1998) of the cervine family. White-tailed deer microsatellites are conserved in bovids (DeWoody et al., 1995), microsatellites are conserved in bovine, ovine and caprine (Kemp et al., 1995) and also conserved between other species of artiodactyls (Engel etal., 1996). The selection of primer pairs for this study then focused on known primers that amplify bovine, ovine and cervine DNA. Dot Blot Procedure The first PCR based genetic typing kit of the HLA-DQ alpha locus using dot blot methodology for detection of sequence polymorphisms was in 1986 (Saiki et al., 1986), with the first use in a criminal case in Pennsylvania in 1986, Pennsylvania v Pestinikis (Blake et al., 1992). PCR amplification of the HLA-DQ alpha locus and detection of point mutations in the different allelic forms using dot blot procedures and allele-specific oligonucleotide (ASO) probes (Saiki et al., 1986; Erlich et al., 1986) has made the transition from RF LP to PCR a simpler technology for detecting polymorphisms. Hybridization, under highly stringent conditions, of a complementary DNA probe to the amplified sequence bound to the membrane is indicated by a color reaction as a spot on the membrane for that particular allele. The presence of a spot is indicative of perfect complementarity in DNA sequence between probe and target DNA thus, identifying the allele present. Reverse dot blot procedure was developed to simultaneously hybridize the 31 DNA procet al.. 1‘} preser RAP! A ampli: This t: oligo 70 pe to 0p; regior be am are ur sePar; P01}‘n me]: and s} SeQU‘e al., 1. for Ur dlDUc DNA to the immobilized ASO probes on the nylon membranes (Saiki er al., 1989). This procedure identifies six different alleles and twenty-one possible genotypes (Helmuth et al., 1990). This method is simple and very time efficient because it will detect the allele present in a single hybridization step by a simple colorimetric reaction. RAPD Technique Another segregating dominant genetic marker amplified by PCR is called random amplified polymorphic DNA (RAPD) and was developed by Williams et al. in 1990. This technique involves the amplification of genomic DNA using an arbitrary oligonucleotide primer, usually nine or ten bases long with a GC content between 50 and 70 percent. This primer binds to homologous sites in genomic DNA and when they bind to opposite DNA strands, which are relatively close to each other, amplification of the region between them will occur. Several sites, randomly distributed in the genome, will be amplified by the oligonucleotide primers thus, creating discrete DNA products which are unable to distinguish a heterozygote from a homozygote. PCR fragments are separated by gel electrophoresis and detected by one of several methods to identify polymorphisms between individuals and generate a genomic profile of PCR products (Welsh and McClelland, 1990). RAPD’s were used to detect genetic diversity in cattle and sheep (Kantanen et al., 1995) and these species show homology with cervids. DNA sequences of RAPD fragments showed high sequence similarity in artiodactyls (Kostia et al., 1996). This allows for characterization of the fragments from one species to another for use in genome mapping of these markers in closely related species, similar to dinucleotide microsatellite homology among closely related species (Moore et al., 1991). 32 den: subs samt tflect prod cfiect dfife confl #1 by P( genor g¢nor Iande (CA).- “'ebe SSCP Technique Single-stranded conformational polymorphisms (SSCP) are also amplified by PCR and are variations in the mobility of single-stranded DNA fragments under non- denaturing conditions. This method of detecting sequence changes, including single base substitutions, is based on the fact that DNA fragments with different sequences but the same length will migrate differently during non-denaturing polyacrylamide gel electrophoresis (Orita et al., 1989a). DNA, in an area of interest, is amplified under standard PCR conditions using 5’ end labeled oligonucleotide primers synthesizing products in the range of 150-250 base pairs (Tuggle, 1994). Fragments generated are electrophoresed and polymorphisms are detected between individuals based on differences in band migration. Single-stranded DNA fragments form folded conformations that are sequence specific and are stabilized by intrastrand interactions (Orita et al., 1989b). Microsatellite Description Microsatellites are excellent codominant genetic markers, amenable to amplification by PCR, due to their high variability, high abundance and widespread distribution in the genome (Weber and May, 1989). Microsatellites are regions of DNA consisting of simple sequence motifs repeated in tandem and abundantly scattered throughout the genome. They are found in many eukaryotic genomes (Tautz and Renz, 1984) and all mammalian genomes so far examined (Stallings er al., 1991). These motifs can be tandem arrays of di-, tri-, tetra-, or pentanucleotides, with dinucleotide motifs such as the (CA)n repeat being the most abundant in humans (Harnada et al., 1982; Beckmann and Weber, 1992) and are also found to be in high abundance in most mammals such as 33 white-tailed deer (DeWoody et al., 1995). The first locus to be tested for microsatellite polymorphisms using PCR was in an intron in the hmnan cardiac muscle actin gene containing variable numbers of the tandem dinucleotide (TG)n repeats, resulting in twelve different allelic fiagments (Litt and Luty, 1989). Microsatellite markers exhibit variability based on the number of repeats that are in tandem at a locus and in the repeat sequence type (Tautz, 1989). Microsatellites can be classified into one of three categories: perfect repeat sequences, imperfect repeat sequences or compound repeat sequences (Weber, 1990). Perfect repeat sequences have no interruptions in the repeat sequence, imperfect repeat sequences have a few scattered single repeat interruptions of a different sequence in the repeat sequence and compound repeat sequences have a tandemly repeated sequence of a different sequence within the repeat sequence. Microsatellite length variations are thought to arise from either unequal exchange during mitosis between sister chromatids or during meiosis between homologous chromosomes or by DNA slippage during replication of the lagging strand (Schlotterer and Tautz, 1992). Microsatellites are presently considered functionless because many of them lie ' outside genes in 3’ or 5’ untranslated regions and roughly ten percent are located in introns between genes. Research suggests that they may facilitate the production rate of proteins according to David G. King of Southern Illinois University, may play a role in forming left-handed conformation (Z-DNA) (Hamada et al., 1982), may regulate transcription (Hamada et al., 1984), may play a role in recombination (Pardue et al., 1987) or may facilitate the pairing of chromatids during mitosis. The positioning of microsatellites in noncoding regions of heterochromatin is forgiving of a high mutation 34 rate. 1 vanai tioni aneni 1999 rnarkt genet Ilarl ‘r. aHoz} thatis not pa notof protei inhen EEneh diagn Ihese rate, estimated to be approximately between 10“ and 10", leading to extensive allelic variation (Saiki et al., 1988). “ . . . it is 10,000 times more likely to gain or lose a repeat from one generation to the next than a gene such as the one responsible for sickle cell anemia is to undergo the single-base mutation leading to that disease” (Moxon and Wills, 1999). This variability of microsatellites, along with their existence as codominant markers following Mendelian inheritance of alleles gives an excellent reason to use these genetic markers for individual identification. Marker Requirements A marker is a genetic unit (gene, microsatellite, minisatellite, restriction site, allozyme, etc.) within the genome that can be followed from generation to generation, that is, from both parents to their offspring. If a region of interest within the genome does not pass from each parent to their offspring, then Mendelian inheritance of that region is not observed and can not be used as a marker to identify individuals. All DNA, whether protein coding or having no sequence dependent function, follows the same rules of inheritance. Mendelian inheritance allows scientists to perform numerous applications: genetic mapping and linkage studies, mapping disease loci, paternity testing, medical diagnostics, pedigree analysis, individual identification, anthropological, evolutionary and population studies and the list goes on. In order for a genetic marker to be useful for these purposes it must follow Mendel’s laws of inheritance. The principle of segregation states that each of the two alleles, one from each homologous chromosome segregate so that the offspring has an equal chance of obtaining either allele from both parents. The principle of independent assortment states that these alleles segregate independently of other alleles at different loci. Individual identification requires population allele 35 freqm that ii for id infor. lengt‘ talc: diver indit that l inher dEIei iden marl frequencies of unlinked polymorphic genetic markers following Mendelian inheritance so that the power of discrimination can be achieved and utilized in forensic applications. The use of- genetic markers for characterizing genetic variability between individuals for identification purposes relies on several factors. The nature of the marker must be informative by showing measurable polymorphisms either in DNA sequence or DNA length between individuals. The polymorphism information content (PIC) of a marker is calculated from allele frequencies in the population. Markers must exhibit genetic diversity among the population, that is, high allelic variation in order to distinguish individuals from each other. The marker must show Mendelian inheritance of the alleles, that is, one allele from each parent must be present in the offspring and should be inherited independently of any other markers being analyzed simultaneously. Lastly, markers that can be easily genotyped will be the marker of choice for individual identification. Selection of Marker, Technique and Method of Detection The selection of the most appropriate molecular marker, molecular technique and detection system for answering a particular question, for example in this study individual identification, is based on several important considerations. The informativeness of the marker, the speed, ease and reliability of the method, costs of the materials, the quality and quantity of DNA, the amount of sequence information required to perform the technique, the dominance of the marker, the detection system and the ease of analysis and interpretation of results, will all be compared between various markers and methodologies. They all have their strong points and weaknesses depending on the results desired and the informativeness needed to answer the desired question. 36 T qualit to resr the cn due to marke requir degrat to the Where Single {Pena suitat using by “.3111.- TElati asP SCVQ The first consideration that might limit the choice of method is how much and what quality of DNA can be obtained from the type of samples at hand. Forensic samples tend to result in a limited quantity of DNA due to the often minimal amount of evidence left at the crime scene. The quality of DNA is usually poor, as samples are partially degraded due to environmental insults. Based on these initial limitations, one may rule out a marker system based on RFLP and Southern hybridization because this technique requires micrograrn quantities of DNA of very high quality. Using RFLP on low quantity degraded DNA will lead to ambiguous results which will be very difficult to interpret due to the presence of extra fragments in the already complex band pattern. In paternity cases where non-degraded samples are readily available, RFLP is an excellent choice using single or multi-locus minisatellite probes where Mendelian inheritance is maintained (Pena and Chakraborty, 1994). PCR amplification of small fragments using any of the suitable systems (RAPD, SSCP, STR) would be the favored choice to resolve questions using minute quantities (nanogram amounts) of degraded DNA (Lorente et al., 1997). The ease and amount of time required to perform a technique and the length of time before obtaining the results are major concerns in this fast-paced society where results are wanted yesterday and for a competitive price. Traditional serologic methods and dot blot assays are the easiest to perform in a short amount of time, usually a few hours, with relatively low cost considering identification kits are manufactured by companies, such as, Perkin-Elmer and Cetus Corporation which do not require expensive detection apparatus. RF LP using Southern hybridization, on the other hand, is very labor intensive requiring restriction digests, gel electrophoresis, Southern transfer, probe hybridization, several washes and film exposure, with results generated in about a week or more. The 37 COST (7 chem requir hybn. Then if rad long 31an Ihror cost of the materials is high for enzymes, membranes, x-ray film and the radioactive or chemiluminescent detection of polymorphisms, to say nothing of the highly skilled labor required to perform the technique; PCR based methods are easy to perform with no hybridizations and results are obtained in a few hours utilizing minimal technologist time. The major cost is in the initial purchase of a PCR machine and a detection system, which can be quite high. The continual purchase of PCR reagents and oligonucleotide primers, if radioactively or fluorescently labeled, is expensive but spreading the cost over several hundred assays reduces the per assay cost. Detection systems can range from simple and inexpensive ethidium bromide, silver staining or colorimetric to the more expensive chemiluminescent, radioactive or fluorescent detection of polymorphisms. The advantages of using ethidium bromide, silver staining or colorimetric are the cost of materials and the ease of use. The disadvantage of using radioactivity is the isotopic component, while the benefit of using chemiluminescence or fluorescence is that they are non-isotopic, safer to use and have a longer shelf life than radioisotopes. Fluorescent detection allows for simultaneous amplification and detection of three or more loci, which greatly increases the speed and throughput of samples with an increase in sensitivity compared to other systems (Lins et aL,1996) The informativeness of the marker along with the dominance or codominance characteristic are very important considerations when choosing the most appropriate marker for the job. Allozyme markers are not very informative because they represent too few loci and exhibit too little variation. RF LP’s probed with minisatellites are at the other end of the marker spectrum, with hypervariability across many loci. Multi-locus 38 minisatellite RFLP’s are extremely informative because they result in numerous bands representing a unique individual-specific DNA profile, except in identical twins. The disadvantage of multi-locus markers is that they are dominant markers only, with some genetic information possibly being lost or hidden due to the absence of a restriction site. Single-locus minisatellite or cDNA probe is not very informative because a single locus will only result in one or two alleles depending on the individual’s zygosity. They are codominant markers with both alleles being represented, one band for homozygous individuals and two bands for heterozygous individuals. The use of several unlinked single-locus probes will increase the informativeness of the technique. In fact this was the first accepted use of DNA testing in forensics. RAPD’s are dominant markers with polymorphic bands scored as present or absent. The informativeness of this marker is limited because a heterozygous individual cannot be distinguished from a homozygous individual. SSCP is considered a codominant marker, with the ability to detect heterozygotes and with limited informativeness because only a single base pair mutation is being detected and only if the substitution changes the mobility of the DNA fragment. Microsatellites are also codominant markers and are extremely informative based on the high degree of polymorphism present among individuals. The amount of sequence information required prior to performing the technique is a matter of concern if money and time are an issue when choosing the method of analysis. RFLP using Southern hybridization does not require actual sequence information, but it does require known gene (cDNA) probe information, though this information may not have to be regenerated for each species tested. The use of a previously identified gene as a probe eliminates the time and money required to obtain species-specific sequence 39 information. Multi-locus probes for DNA typing are VNTR’s which are not necessarily species-specific, but require sequence information, with no effort and cost to attain. PCR derived systems require exact sequence information for the development of oligonucleotide primers with high cost and effort to obtain. However, comparative mapping utilizing primer pairs from closely related species can be both economical and time efficient when there is conservation of heterologous primer pairs between species (Moore et al., 1991). RAPD’s, on the other hand, do not require any sequence information due to the arbitrary generation of oligonucleotide primer pairs used in the technique. Interpretation and analysis of results can be ambiguous or straightforward depending on which marker and detection method are used. Traditional serologic procedures using agglutination of antigens and staining of allozymes with colored dyes, along with the colorimetric detection in dot blot assays are very straightforward analyses. The presence of a reaction taking place, detected by agglutination, staining or color development, is very unambiguous, that is, present or absent. Analysis of RFLP’s when a single-locus probe is used is straightforward, depending only on the presence of one or two bands of a specific size. Use of a multi-locus probe can lead to a very problematic analysis. The banding pattern can be complex due to the ntunber of fragments present, the possibility of extra fragments from slightly degraded samples or the sharing of bands between loci can make interpretation of the data challenging. The use of size standards in adjacent lanes of an RFLP gel can result in estimates of allele sizes, usually with errors of 2-5 bp. RAPD’s can also be difficult to interpret because of the presence of many bands, some of which could be similarly sized alleles from different loci and some of which are artifactual 40 - Fin based on the sensitivity of the assay to the PCR conditions. SSCP analysis can be difficult if the running conditions are not optimized because slight changes can effect the mobilities of single-stranded DNA moleCules leading to unpredictable results. Lastly, microsatellites can be easy to score if the tandem repeat is not a dinucleotide motif because these alleles show a characteristic stutter band due to the slippage of the polymerase during extension of the PCR product (Hauge and Litt, 1993; Murray et al., 1993). The stutter usually creates a band two and four base pairs smaller in size than the original fragment size, creating ambiguities in calling a true heterozygote with alleles differing by a single repeat unit from a homozygote with a strong stutter band. However, the presence of these characteristic stutter bands is beneficial in separating true alleles from background noise. Fluorescent detection of microsatellite alleles makes calling the sizes of fragments more accurate because of the use of an internal size standard in each lane of the gel, thus, correcting for lane to lane and gel to gel variability. The information presented here, summarized below in Table l, and in this literature review is the basis for the selection of using ubiquitous, microsatellite markers, fluorescent detection and PCR technology to achieve the goal of this research project. The objective of this study is to develop a practical, economical, time-efficient DNA typing system for wildlife forensic scientists to utilize for individualizing forensic evidence related to Michigan white-tailed deer. 41 Table 1. Comparison of markers, techniques and methods of detection. Marker/Technique Characteristic serologic/dot blot/ RAPD,SSCP, RFLP/Southern dot blot-PCR STR/PCR hybridization quality of relatively fresh samples low molecular high molecular sample dot blot-PCR: low weight DNA weight DNA molecular weight DNA quantity of minimal nanogram microgram sample amounts amounts amounts turn around time few hours one or two several days to for results days a week or more technologist time minimal time minimal time labor intensive required to easiest to perform easy to perform requires a skilled perform technique technologist commercial kits initial cost: cost of can be expensive expensive cost: materials overall: continual cost: very high very cheap inexpensive least informative most informative most informative Informativenecs dot blot-PCR: when using STR’s, when using multi- of marker very informative otherwise very locus probes limited marker RAPD’s: dominant single-locus: characteristic dominant SSCP: codominant codominant STR’s: codominant multi-locus: dominant sequence none RAPD’s: none single-locus: none information dot blot-PCR: SSCP: complete multi-locus: complete requirement complete STR’s: complete RAPD’s and SSCP: single-locus: interpretation straightforward ambiguous straightforward of results STR’s : multi-locus: unambiguous ambiguous Detection System silver staining chemiluminescent ethidium bromide radioisotope fluorescent colorimetric high sensitivity safer to use advantage of simple to use clear interpretation easier to dispose of detection system inexpensive of results longer shelf-life high sensitivity disadvantage of limited sensitivity use of detection system ethidium bromide is a radioactivity expensive carcinogen 42 METHODOLOGY This study involved using random samples of the population of Michigan white- tailed deer to establish. a database. Tissue samples were collected from deer in all 83 counties in Michigan by Tim Tesmer, Jessie Marcus, Ron Southwick and Dr. Paul Coussens. The Department of Natural Resources (DNR) and the US. Fish and Wildlife Service jointly funded the project in 1994. Hunters were asked at DNR checkpoints to voluntarily allow these samples to be collected. The deer’s age, sex, county and deer management unit (DMU) were documented on the collection tube for each sample collected. Muscle tissue was collected and samples were placed in polypropylene tubes with caps and kept in a -20° C freezer until needed for DNA extraction. To date approximately 1200 deer tissue samples have been collected with only a few counties not totally represented. Bay, Clinton, Macomb and Sanilac counties have only one collected sample, while Lapeer has four and Keweenaw has none. These counties will not be fully represented in the database on the basis of five random deer samples per county unless additional samples are collected prior to the completion of this study. Tissues for many of the counties had DNA already extracted by Ms. Jean Robertson using a standard protein digestion, phenol/chloroform extraction and ethanol precipitation procedure (Sambrook et al., 1989). The quality and quantity of these DNA samples when initially measured seemed to be good on paper however, many of these DNA samples have since degraded. The quality of these DNA’s was tested on a 0.8% agarose gel (SeaKem LE Agarose from F MC) (see Appendix A for gel preparation). Good quality 43 used for database acquisition, while the remainder of the samples, about 185, were re- extracted using a slightly modified procedure (see Appendix B for extraction procedure). The reason for this modification was to obtain the highest quality and quantity DNA possible from these tissues. DNA samples used for the initial screening of the eight fluorescently labeled microsatellite markers (see Table 2 for details) along with the 425 samples necessary for the database were selected at random. DNA samples with low quantitation (less than 15 ng/ul) and/or low absorbance ratio, 260/280, (less than 1.5) were ultimately excluded for failure to provide acceptable data. Twenty samples were randomly selected from the approximate 1200 deer samples collected in 1994, measured on a Milton Roy Spectronic Genesys 5 spectrophotometer for an estimate of DNA quality and on a Hoefer TKO 100 fluorometer for DNA concentration and adjusted to be approximately 30 ng/ul. The 425 samples for the database, five from each county and two islands, were processed in the same manner. All samples were thus quantitatively standardized prior to performing polymerase chain reactions. 44 .3335 2: 33.8 3 .3533 028.922: 9.0.3 22. 5 9.8.52 a geomoeozo $2.30 50550055055058 380.30 2.5 9.75 x5 05255550555355 .usawom 380.50 <29 830 e 058.820 96:5 0055050555050 2:8 more: 5: 00842505500050: 8.3. .585 mzmo «N 258525 €280 0<5<5e50<5U DZQHXH ZO—Hé:h > > _ 5m cam as; one; W ”M”: fl Nb: v / I can :28 8:6 8:.» 3.3: W 35: I 8:6: \ 81:: \ .3 83: 8:85 38:3..— I 8::aw—IEQN555awbemVMNa 54 the 260nm/280nm absorbance ratio from the absorbance readings at wavelengths 260nm and 280nm. The ratio provides an estimate of the purity of the DNA. PCR OPTIMIZATION OF EIGHT MARKERS Two samples along with negative and positive controls were selected and used for initial PCR optimization of the eight markers. Figures 6 through 11 show the eight markers optimized and electrophoresed on agarose gels along with known size standards in external lanes. The presence of a single band in the approximate fragment size range based on species homology listed in Table 2 indicated that the PCR conditions were optimally amplifying the locus of interest. The absence of a band for the negative control indicates no DNA contamination and no PCR artifacts present in the size range of interest. INITIAL SCREENING OF MARKERS WITH TWENTY SAMPLES Two of the most important aspects of DNA markers are heterozygosity and diversity. A goal of this study was to identify a set of markers with high heterozygosity and numerous possible alleles in the test population. The next set of results were obtained to determine the extent of genetic diversity and to select those markers exhibiting high polymorphism among the species using twenty random samples containing high quality DNA. These samples were amplified with fluorescent primers using each marker’s optimal PCR conditions and electrophoresed on an ABI 377XL DNA Sequencer. The initial screening data for the twenty samples for each marker along with the number of heterozygous samples, percent heterozygous, number of different alleles, the allele size range and polymorphic information content (PIC) are presented in Table 5. The PIC value was calculated using the formulation of Botstein et al. (1980) and is indicative of a 55 1234567—Lanes — Loading Wells 501 bp 489 404 331 242 190 147 l l l l l 0 67 Alleles —> . I /\\\\\\\ | / | Positive Negative Hpa II Figure 6. Agarose gel of marker: JP15. 1 2 3 4 5 — Lanes Loading Wells 587 bp 458 434 298 267 257 174 102 so Alleles —> A\\\\ I / I / | Positive Negative Hae 111 Figure 7. Agarose gel of marker: CSN3. 56 123456 789—Lanes ‘ ' — Loading Wells 1 d 501 bp 489 404 331 242 190 147 \ 111 7 110 ///// 4— Alleles — Primer Dimer | / | Hpa 11 Positive Negative Figure 8. Agarose gel of marker: IRBP2. 1 2 3 4 5 6 7 8 91011121314151617181920 — Lanes 2, L".- ¢§1%'fi'“wk°‘ 4'13 '3‘; 3L"; 55’? I“. 4.5 '. 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EPZDOU .8383. .39.: 82:52 wfim: €8.38 Ea? ..o gm .m< :a 8?... 9:2. 8:2. ... $.39: w:.=oo.:um 3...... .m 03a... 59 marker’s ability to differentiate individuals. Heterozygosity was calculated by dividing the number of heterozygous individuals by the total number of individuals. Heterozygosity of the eight markers screened on twenty random deer samples is graphically represented in Figure 12. Marker loci CRFA, OBCAM, IRBP2 and IGF] displayed a minimum 70% heterozygosity and appeared to be highly polymorphic. These markers were selected for use in analysis of database samples. Figure 13 shows a graphical representation of the degree of polymorphism for the three selected markers CRFA, OBCAM and IGF]. These three markers could be amplified under similar PCR conditions and thermocycler temperature programming (multiplexed). The ability to multiplex was considered critical since the final test needed to conserve cost as well as time. 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PCR does not require the highest quality or quantity DNA, however, amplification of the markers in this study was affected by these two variables. Samples exhibiting low quality and/or low quantity DNA often lead to unpredictable and unreliable results. Samples of this nature, containing either minimal amount of intact target DNA and flanking primer sites or very few copies of the target sequence, resulted in weak amplification products which were unable to be scored confidently. Problems were encountered in accurately scoring the alleles present for many samples with optical densities less than 1.5 and/or concentrations less than 15 ng/ul. Therefore, DNA was extracted from many deer samples in order to alleviate any scoring discrepancies. Freshly extracted DNA samples resulted in high molecular weight DNA facilitating unambiguous scoring of alleles. The most successful method for extracting these tissue samples, resulting in the highest quality and quantity of DNA, was Proteinase K protein digestion using Qiagen lysis Buffer ATL followed by phenol/chloroform extraction and ethanol precipitation. Digestion with Proteinase K instead of Protease and the use of a commercial lysis buffer instead of a homemade lysis buffer yielded higher quality DNA. Also, the use of the Qiagen QIAamp tissue kit using spin columns resulted in low quality and quantity DNA, (data not shown). The options tried in this research project are not all inclusive of the 107 many different possible combinations of tissue extraction materials and methods that can be used. However, this combination clearly worked the best for the needs of this research project. . Along with the benefits of using PCR-based systems for individualizing forensic evidence, such as extreme sensitivity and specificity, simple and quick to perform, speed of amplification and the applicability to small quantities of degraded samples, comes the concern of PCR misincorporation rate, error and contamination. The issue of contamination was not observed because samples were handled under good laboratory practices and when they were genotyped, the results clearly comprised of no more than one or two alleles for each sample. The appearance of shadow bands, two nucleotides shorter than the correct allele, is a resultant error on the part of the polymerase due to slippage during chain elongation. These bands were present for all the markers used in this study and their appearance did not affect the scoring of the correct alleles. To the contrary, having these shadow bands made the identification of alleles easier whenever the allele intensity was weak. The other source of error by the polymerase is the addition of a nontemplated nucleotide, usually an A, to the 3’ end of the PCR product. This situation is marker specific with some markers being affected and some not at all, as was the case in this study. All the markers were not affected by this polymerase error, except the marker JP23, which resulted in both products, the true allele and the true allele + A. Correction for this error was to facilitate the nontemplated nucleotide addition by adding an additional five minute final extension step to the end of the thermocycler program in order to favor the production of the allele + A product. The misincorporation rate of T aq polymerase used in this study is approximately 10“ nucleotides/cycle (Saiki et al., 1988). 108 This error rate is minimal and would only be detectable by electrophoretically visible bands if the misincorporation was during the beginning cycles of PCR, with very limited amount of template and affecting many copies (Saiki et al., 1988). Misincorporated nucleotides will not be detected because the visible product on the gel will be the majority of the amplified product. The incorporation of an incorrect nucleotide will not change the length of the fragment. Eight microsatellite markers were initially used to assess genetic diversity among twenty random deer DNA samples resulting in four highly polymorphic loci. CRF A, OBCAM, IGF 1 and IRBP2 all exhibit high heterozygosity and high polymorphic information content (PIC) and therefore show genetic variability. The calculated PIC value, according to the formulation of Botstein et al. (1980), is an indicator of a genetic markers’ ability to distinguish between individuals and was used in selecting markers for database acquisition. A marker with a high PIC value is indicative of numerous allelic variants in the sampled population, and this makes it less likely that two random individuals will have the same alleles. The presence of several different alleles occurring in heterozygous individuals allows for the greatest power of discrimination, and the larger the excluded population, the greater the weight of the evidence. While CSN3, FCB304 and JPIS do not show a high enough degree of variability to have high discriminatory potential, JP23 has a high PIC value, but very low heterozygosity and thus could be an informative marker if combined-with other polymorphic markers. The low heterozygosity with a high PIC value could be due to the presence of nonamplifying or ‘null’ alleles, which would result in mistyping a heterozygous individual as a homozygous individual. This was probably due to the loss of complementarity in 109 nucleotide sequence or mutation at the primer binding sites (Callen et al., 1993; Pemberton et al. , 1995), or too few intact copies of target DNA at that locus resulting in weak amplification of alleles, unable to be scored accurately. This marker, JP23, was not selected for database acquisition because many samples had amplification problems resulting in no products. Amplification problems ofien occur when using primers derived from species other than the one which it was cloned. Marker selection criteria for database acquisition focused on the development of a practical, economical, time-efficient DNA typing system for individualizing forensic evidence. This entails being able to multiplex several markers in one PCR and run them simultaneously in one electrophoretic lane of an ABI 377 DNA Sequencer in order to save on both money and time. Three of the four highly polymorphic markers were selected for database acquisition based on these criteria: OBCAM, CRFA and IGF 1. The marker IRBP2 was eliminated because the annealing temperature was five degrees lower than the other three markers and therefore, could not be multiplexed together. The three selected markers have high heterozygosities, high PIC values and a high number of allelic variants making them the obvious choices for the development of a marker panel with a potentially high power of discrimination. These markers also satisfy the quest for one multiplex PCR to be analyzed simultaneously in a single lane of a polyacrylamide gel. Optimal PCR conditions for all three markers were performed using the same amounts of each PCR component and the same annealing temperature of the thermocycler running conditions. The allele size ranges of these markers do not overlap, thus allowing them to be run simultaneously in one lane of a polyacrylamide gel. Each 110 marker is also separated by a different fluorescent dye, which eliminates any miscalling of alleles between each marker. The use of heterologous primer sequences for these markers derived from bovine and ovine sources are homologous in cervines. Many studies have shown that within the order Artiodactyla, primer pairs from other species can amplify homologous products in related species of deer (DeWoody et al., 1995; Engel et al., 1996; Roed, 1998; Slate et al., 1998; Kuhn et al., 1996; Pepin et al., 1995; O’Connell and Denome, 1999; Talbot et al., 1996). This conservation of heterologous PCR primer pairs between related species has facilitated the development of this microsatellite marker system for individual identification by saving time and money that would have been required for cervid primer development. Primer pairs for the loci OBCAM and IGF] were derived from bovine DNA and CRF A was derived from ovine DNA. These microsatellite loci must follow Mendel’s laws of inheritance in order to be considered genetic markers. Moore et al. in 1992 proved Mendel’s principle of segregation for both OBCAM and CRF A by showing that in one bovine family, one allele from each parent was present in the five offspring sampled. Adams & Maddox in 1994 proved Mendelian inheritance of the IGF] microsatellite in two two-generation ovine families. This study analyzed nine two- generation white-tailed deer families of a captive deer population from Savannah River, Georgia, and supplied by Dr. Jerry L. Ruth with the Fish and Wildlife Service of the United States Department of the Interior. The results from these families proved Mendelian inheritance of all three microsatellite markers used in this study. Mendel’s principle of independent assortment states that alleles for one marker segregate 111 independently of alleles for the other loci being analyzed simultaneously. The location of IGF 1 is on bovine chromosome 5, OBCAM is on 29 and CRF A is on ovine chromosome 9. As all three markers are on separate chromosomes for the related species bovine and ovine, which verifies independent assortment of alleles, and with the proven homology between species of Artiodactyls, the same assumption was made for cervids. There is no evidence of association of alleles among the three loci selected for database acquisition. The alleles for each marker show no signs of being linked to each other. These loci are inherited independently, which allows for the product rule to be applied when calculating multiple loci profiles from the observed allele frequencies under the assumption of Hardy-Weinberg equilibrium. To address the issue of deviations from random mating in the sampled population of Michigan white-tailed deer, tests for Hardy-Weinberg equilibrium (HWE) were conducted. HWE was statistically tested for the sampled population in this study by using the Fisher Exact test, which tests for exactness to HWE by comparing the observed genotypic frequencies with those expected under HWE. That is, Hardy-Weinberg law states that genotypic frequencies will remain constant from generation to generation as long as the following assumptions remain true: the absence of selection, migration and mutation along with the continuation of random mating (King and Stansfield, 1990). The Fisher Exact test was selected because of the possibility of rare alleles occurring in very low frequency in the sampled population, and these small numbers will have very little effect on other tests performed at the 5% level of significance even in large sampled populations. The Fisher Exact test can identify significant differences between low frequency alleles and genotypes. 112 The P-values for the three markers for the sampled population along with the four separate regions of the population all show no significant deviation from HWE at the Bonferroni corrected significance level of P = 0.004 (Table 9). This probability or significance level measures the probability of a type I error, that is, the probability of falsely rejecting a true hypothesis. However, the marker CRF A appears to approach significance for the overall population and especially for the North East Lower Michigan region, which indicates the possibility of population substructure or inbreeding in that region. This could be particularly true for this region because of the feeding arrangement set up by hunters for deer in the area where tuberculosis was recently detected at higher levels. The microsatellite marker CRFA is associated with the gene corticotropin- releasing factor, which is involved in the body’s response to stress and has an effect on the immune system. The incidence of tuberculosis in this region may therefore have resulted in selection pressure at that locus. This makes sense because the deer in the North East Lower Michigan were exposed to tuberculosis around the time the tissue samples were collected in 1994 and CRF A was the only marker of the three approaching significant deviation from HWE. The deer contained to this area, because of the excellent feeding arrangement, would thus constantly inbreed amongst each other and result in homozygote excess at the CRFA locus. This excess of homozygotes or deficiency of heterozygotes can readily be seen in Table 8 for CRF A, 81% observed heterozygosity compared to 87% expected heterozygosity. Table 11 shows that much of this excess of homozygotes is attributed to the Northern part of the lower peninsula with Table 13 showing that the North East region is causing much of the deviation from the expected heterozygosity. There are obvious deviations in observed heterozygosity compared to 113 HWE expectations, but only for CRF A. Another possible reason for the deviations could be the migration of deer northward because of fires in the southern part of the state years ago, causing the deer to be in close proximity to each other and to breed among siblings. The scope of this study cannot fully address the reasons for the homozygote excess for the marker CRF A in the population without additional testing however, this study did address the question of whether the population is consistent with HWE. The results (Table 9) were that HWE holds true at each locus in each region of Michigan and the state as a population. This means that the sampled population exists in equilibrium to the expectations of the Hardy-Weinberg law and genotype frequencies can be reliably estimated from allele frequency data. Further continuation of microsatellite marker testing on Michigan white-tailed deer can be based on random mating and statistical analyses assuming HWE can be performed. If not in HWE, then future testing would have to use different statistical analyses to account for disequilibrium. An overall chi-square (X2) was also calculated along with the Fisher Exact test by the GENEPOP V3.1d software program resulting in the same insignificant results when comparing all three markers for each region of Michigan for deviations from HWE at the same P = 0.004 (Table 9). These differences have a high probability of occurring by chance and are statistically insignificant, thus no deviation from Hardy-Weinberg equilibrium. A goodness of fit chi-square test means that the observed results do not differ significantly from what would be expected. This test aims to test the hypothesis that there is close agreement between what was observed in the sampled population and the expectations of HWE. These two statistical tests for Hardy-Weinberg equilibrium are not good indicators of population substructure therefore, the results cannot imply the 114 absence of substructure. To detect substructure, the population of Michigan was subdivided into selected regions and statistical analyses were performed on them. However, the best and most accurate way to detect substructuring is to sample the individual subgroups and observe the genotype frequencies between them. To address this issue of a possible substructured population of Michigan white-tailed deer sampled in this study, tests for inter-population variance in allele frequency were conducted. That is, if there are differences in allele frequencies between the four regions in Michigan, then population substructuring exists. Even though tests for random mating under HWE were insignificant for each marker within each region of Michigan, testing for differences between these regions using the F -statistic (F s,) to estimate spatial heterogeneity in allele frequencies was conducted. Table 15 lists the allele frequencies for each marker in each region along with the calculated F s, value for each marker analyzed across the four regions. The calculated chi-square value measures the degree of significance of the FS, value, taking into account the large sample size of approximately 450 samples. The F s, values for the three markers across the four regions were low but statistically significant based on the chi-square values. This means that there is not a lot of variance between regions for allele frequency, but considering the large amount of samples, the FS, values for each marker become statistically significant after the chi- square test. Chi—square values greater than the level of significance (P = 0.004) afier Bonferroni correction rejects the hypothesis of population homogeneity (F s, = 0) in allele frequencies and thus, indicates spatial heterogeneity within the sampled population of Michigan white-tailed deer. 115 Table 16 shows pair-wise comparisons between all regions for allelic frequency differences. The chi-square values are statistically significant at P = 0.0028 after Bonferroni correction. All combinations are significant except when comparing the three regions to the North East Lower region. The marker IGF 1 for North West Lower and Southern Lower and CRF A for the Upper Peninsula are insignificant when compared to the allele frequencies in the North East Lower. Overall, allele frequencies differ between the four regions in Michigan, also known as the Wahlund effect because the population consists of a number of subpopulations having different allelic frequencies. Accurate calculations of probability estimates for linking forensic evidence to a suspect should apply the appropriate allele frequencies for the region of Michigan where the crime was committed. In the event of a match, the calculated probability using allele frequencies at the population level compared to using subpopulation frequencies, will not have a significant effect on the value of the match; the match will still be a match whether the probability is one in a million or one in ten million. A match between evidence at the crime scene and evidence on the suspect or in the suspect’s possession clearly places the suspect at the crime scene. The purpose of this research project was to develop a practical, economical, time- efficient DNA typing system for wildlife forensic scientists to utilize for individualizing forensic evidence. This DNA typing system has the ability to match two physically separated deer samples, thus placing the suspect at the scene of the crime and providing a powerful law enforcement tool for wildlife officials. This research project has developed a microsatellite marker panel which can be multiplexed in one PCR and run simultaneously in one electrophoretic lane of a DNA sequencer. This panel of markers 116 along with the application of molecular biology techniques will save forensic wildlife scientists time and money by assisting them in achieving their goal of enforcing wildlife laws. This DNA typing system has a power of discrimination of 0.9998 or 99.98 %, and in the event of matching two physically separated samples with forty-six possible allelic variants, accuracy in calculating probability estimates can be achieved, even when the population is substructured. Forensic wildlife scientists can also use this system for parentage determinations, which was validated by the testing of the nine pedigreed families in this study, and distinguishing native white-tailed deer from either imported white-tailed deer or deer from outside the Michigan area. 117 FUTURE DIRECTIONS Recommendations for future improvements to this study for continued research in this area of forensic identification of Michigan white-tailed deer will be discussed. The group of samples used in the present study was limited to five samples per county, representing a random sampling of all Michigan deer, but not a representative sampling from each county. Some counties were underrepresented in the total sampled population of Michigan deer. Therefore, the frequency of alleles will not be an accurate representation of the whole Michigan deer population at large and when calculating the probability of a match, the estimate will not be truly accurate. If more samples were genotyped from each county, the allele frequencies would be exceedingly more accurate and new or rare alleles would arise, thus resulting in higher genetic variability. The goal of any identification system is to obtain all possible genetic variants in the population prior to analyzing evidentiary material. This study resulted in four highly polymorphic markers of which three were ultimately used for database acquisition. The fourth marker, IRBP2, would be an excellent marker to add to the panel for many reasons. The percent heterozygosity, polymorphic information content and number of different alleles is relatively high. The allele size range would allow the marker to be analyzed simultaneously with the other three markers in one lane of a DNA sequencer. The fluorescent tag is HEX and with the neighboring markers in the panel being labeled with F AM and TET, IRBP2 could be used without the risk of any interfering problems. The PCR conditions are compatible with the other three markers, however, the annealing temperature used in the thermocycler 118 temperature programming is 50 degrees instead of 55 degrees as for the developed panel. This means that in order to add this marker to the panel, the length of the oligonucleotide primers would need to be extended an additional few nucleotide bases so that the T m for each primer would be five degrees higher. The addition of an AC to the 3’ end of the primer 5’-GTATGATCACCTTCTATGCTTCC-3’ would raise the T m by approximately six degrees because for every A or T added the T m raises approximately two degrees and for every C or G added the T m raises approximately four degrees. The addition of a CA to the 5’ end of the primer 5’-CCCTAAATACTACCATCTAGAAG-3’ would also raise the Tm by approximately six degrees. In order to have optimal PCR conditions, both primers need to have similar T m’s and a G or C at the 3’ end to anchor the primer so efficient elongation by the polymerase can occur. Other considerations when adding these two bases are sequence complementarity between primers and the formation of secondary structures, so checking these new sequences through an oligonucleotide primer design program prior to synthesizing the primers is highly recommended. These additional bases were selected from adjacent sequence information for the primers for this marker, located in GenBank (Accession # M20748). This sequence information was unavailable during this research project, therefore, not pursued for this microsatellite panel. The addition of this marker along with other polymorphic markers to the developed panel will increase the power of discrimination of the system, thus making it an even more powerful tool for individualizing forensic evidence. A more accurate evaluation of the data presented here for testing the substructure of the population of white-tailed deer in Michigan would take into account other factors 119 when subdividing the deer population in Michigan. For this study, the assessment of the structure of the population was based on dividing the state into four regions based on county lines, which has no relevance biologically. A future study could accurately assess the division of Michigan based on actual geographical barriers, which would be more relevant to answering questions concerning the structuring of the Michigan white-tailed deer population. Wildlife forensic scientists will benefit from the results of this study in several ways. They will have an easy, low cost, reliable scientific test to perform, with results in less than one day, on evidence linking a suspect to a crime when a suspect has been identified. That is, to determine whether the field evidence matches the evidence found in the possession of the suspect with a high degree of probability. This DNA typing system will assist law enforcement officials in convicting poachers by providing critical evidence linking evidentiary samples and parentage testing for illegal importation cases. The mere fact of having the ability to individualize a deer for forensic purposes will hopefully deter the criminal from committing the crime. As for many crimes committed by humans, members in the criminal justice system use deterrence, usually harsh sentencing and high penalties, to decrease the number of committed crimes. However, the effectiveness of this deterrence remains to be seen, but for wildlife law enforcement officers it would be a step in the right direction to minimizing poaching crimes. A future study could evaluate the deterrent effect and the number of convictions based on having this DNA typing system available to wildlife forensic scientists. The data presented in this study has laid the groundwork for several future applications, other than individual identification, of Michigan white-tailed deer. 120 Database acquisition is a necessary requirement for calculating probability estimates for individual identification, and exclusion/inclusion of parentage as well as the determination of the extent of population substructure, genetic diversity, population size and the detrimental effects of inbreeding. Evolutionary and comparative mapping studies between related species can also be accomplished utilizing the accumulated data from this study. The assessment of captive breeding programs for the loss of genetic diversity is another possibility. Identification of relatedness among pedigreed and captive populations, so that the selection of matings will preserve genetic variability in order to maintain the species, can furthermore be accomplished. 121 APPENDIX A 122 10. ll. 12. APPENDIX A AGAROSE GEL PREPARATION PROCEDURE Determine the gel percentage and the number of wells needed. A small gel (15cm x 15cm) requires at least 125 ml of agarose solution. Weigh the correct amount of agarose (SeaKem LE Agarose, Cat # 50007 from FMC) based on the gel percentage and place in a 250 ml Erlenmeyer flask. A 0.8% gel (for genomic DNA) would need 1 g agarose and a 2% gel (for PCR products) would need 2.5 g agarose in 125 ml of IX TBE (Tris/boric acid/EDTA). Prepare a 10X TBE solution with 108 g Tris, 55 g boric acid, 8.3 g EDTA and quantity sufficient to l L with deionized water. Dilute to 1X TBE with deionized water for use in gel preparation and for the electrophoresis buffer. Mix the agarose and 1X TBE in the flask by either heating on top of a heated stir plate or on low in a microwave until the agarose has completely dissolved. Watch the solution in the microwave to prevent the solution from boiling over and becoming a different concentration. Once dissolved, add deionized water to correct for any loss due to evaporation during heating. Let the solution cool until the flask is able to be handled by the hands; too cool will show signs of hardening in the flask and too warm will warp the plastic tray. Ethiditun bromide (Cat # E-8751 from Sigma) is not added to the solution for possible interference with fragment migration. The gel afier electrophoresis is submerged in an ethidium bromide solution in order to detect the fragments. Place appropriate comb(s) into the level gel tray and carefully pour the agarose solution into the tray so as to not introduce bubbles. Allow the gel to stand and harden for at least 30 minutes at room temperature. Quicker hardening may be achieved by placing the tray in the refrigerator. Remove the comb(s) with the gel submerged in the electrophoresis buffer (1X TBE) to prevent tearing the wells. Add enough buffer to the electrophoresis chamber to cover the gel and load the samples into the wells when ready. 123 APPENDIX B 124 APPENDIX B TISSUE EXTRACTION PROCEDURE A. TISSUE DIGESTION 1. Cut 50 mg of frozen tissue into very tiny pieces with a sterile scalpel and place in a 1.5 ml polypropylene tube. 2. Add 250 pl of Qiagen Buffer ATL (Cat # 19076) to each tube. 3. Add 25 ul of 20 mg/ml Proteinase K (Cat # 25530-015 from Gibco BRL) to each tube. 4. Vortex each tube vigorously for 30 seconds until a homogeneous mixture is obtained. 5. Incubate all tubes in a 55° waterbath overnight or until the tissues are lysed completely. B. PHENOL/CHLOROFORM EXTRACTION 1. Add 300 pl of phenolzchloroformzisoamyl alcohol, 25:24:], saturated with lOmM Tris, pH 8.0, lmM EDTA (Cat # 0883-100mL from Amresco) to each tube. 2. Invert the tubes several times to thoroughly mix the contents. 3. Spin the tubes for 5 minutes at full speed (12,000 rpm) in a microcentrifuge. 4. Remove the top aqueous layer (approx. 300 pl) and add to a fresh tube. 5. Repeat steps 1 through 4. 6. Add 300 pl of chloroform (Cat # 9180-03 from J .T. Baker) to each tube. 7. Invert the tubes several times to thoroughly mix the contents. 8. Spin the tubes for 5 minutes at full speed (12,000 rpm) in a microcentrifuge. 9. Remove the top aqueous layer (approx. 300 pl) and add to a fresh tube. 125 10. 10. ll. Perform steps 1-9 in a vented hood due to the carcinogenicity of the chemicals. ETHANOL PRECIPITATION Add 600 pl of 100% ethanol and 30 ul of 3M sodirun acetate to each tube. Invert the tubes several times to fully precipitate the DNA. Spin the tubes for 5 minutes at full speed (12,000 rpm) in a microcentrifuge. Carefully decant the contents of each tube without losing the pelleted DNA at the bottom. Add 1 ml of 70% ethanol to each tube. Invert the tubes several times to thoroughly wash the DNA. Spin the tubes for 5 minutes at full speed (12,000 rpm) in a microcentrifuge. Carefully decant the contents of each tube without losing the pelleted DNA at the bottom. Resuspend the DNA in the tubes, after the DNA has air dried, in 200 pl Tris/EDTA (TE), pH 8.0. This amount should give a yield of approximately 200 ng/ul of high quality DNA with an absorbance ratio (260nm/280nm) in the range of 1.7-1.9. Measure the quantity of DNA in the samples using a fluorometer which yields a measurement of only DNA and not RNA. Measure the quality of DNA in the samples using a spectrophotometer which yields a ratio of both DNA and RNA to protein in the samples. Dilute the DNA samples to approximately 30 ng/ul with deionized water. These samples are now ready for a polymerase chain reaction (PCR). 126 APPENDIX C 127 10. 11. 12. 13. APPENDIX C ACRYLAMIDE GEL PREPARATION PROCEDURE Prepare a 10X Tris/boric acid/EDTA solution (TBE) with 108 g Tris, 55 g boric acid, 8.3 g EDTA and quantity sufficient to 1 L with deionized water. Add 18 g urea (Cat # IB72064 Molecular Biology Grade from Kodak), 5.3 ml 40% Acrylamide/Bis 19:1 (5% C) (Cat # 161-0120 from Biorad), 25 ml deionized water, 5.5 ml 10X TBE and approximately 0.5 g of Amberlite MB-l 50 (Cat # A-5710 from Sigma) to an Erlenmeyer flask. Mix the contents using a magnetic stir bar on a stir plate until dissolved completely. Filter and degas the solution using a 0.45 micron bottle top filter (Cat # 290-3345 from Nalgene). Prepare a 10% ammonium persulfate (Cat # O486-100G-APP from Amresco) solution (APS) with deionized water. Add 250 pl of the 10% APS to the acrylamide solution. Gently swirl the bottle to mix the contents without introducing bubbles. Add 35 ul TEMED (Cat # 161-0800 from Biorad) to the acrylamide solution. Gently swirl the bottle to mix the contents without introducing bubbles. Pour the solution quickly into the clamped ABI 377 DNA sequencing plates without introducing bubbles and before the solution polymerizes. Place the appropriate sized comb into the plate opening and clamp together. Let the gel stand for at least 1 1/2 hours before using to assure complete polymerization. Acrylamide is a neurotoxin so extreme care should be taken and appropriate safety equipment should be worn during the above steps. 128 APPENDIX D 129 APPENDIX D MULTIPLEX PCR PROTOCOL MULTIPLEX PCR COMPONENTS FOR A 25 ul REACTION Pipet 2 ul of each 30 ng/ul DNA sample into separate microcentrifuge tubes. 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