Analysis of genetic markers in forensic DNA samples using the

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Anal. Chem. 1991, 63,2-15

PERSPECTIVE: ANALYTICAL BIOTECHNOLOGY

Analysis of Genetic Markers in Forensic DNA Samples Using the Polymerase Chain Reaction Rebecca Reynolds*J and George Sensabaugh

School of Public Health, Forensic Science Group, University of California, Berkeley, California 94720 Edward Blake

Forensic Science Associates, 3053 Research Drive, Richmond, California 94806

The ablllty to extract and type DNA from forensic evldentlary samples has revolutlonlzed the field of forensk serology. Previously, genetic marker typing was llmlted to the analysis of blood group markers and soluble polymorphic protein markers. Because the number of suitable markers expressed In partlcular flulds and tissues Is relatlvely small, and because mlxtures of flulds cannot be separated for conventional genetlc marker typing, a suspect frequently cannot be Included or excluded as a fluld donor In a case. However, the development of methods to extract DNA from virtually all blological specimens has greatly expanded the potential for lndlvldual Identtfkatlon. 01 partlcular Importance was the ability to extract mlxtures of sperm cells and epithelial cells found in sexual assault cases such that the DNA from the sperm cells could be typed Independently of the DNA from the vlctlm’s epithelial cells. Restriction fragment length polymorphism (RFLP) analysls was the first DNA-based method applied to problems of lndlvldual lndentlflcatlon. This method, while powerful In Its abllity to dtfferentlate Indlvlduals, Is llmlted by the quantity and quallty of DNA required for an unambiguous result and by the amount of time It takes to obtaln a result. Desplte these Ihnltations, several laboratorles are using RFLP analysis successfully for the detectlon of pdymorphlsms In forensic DNA case samples. Whlle the field of forensic serology was belng revolutlonlzed by the prospect of DNA analysis, the field of molecular biology was belng revolutlonked by the Invention of the polymerase chaln reaction (PCR), which ultimately has had an Impact on every area of blologIcal sclence. The PCR DNA amplMcath technology Is Ideally suited for the analysls of forensic DNA samples In that It Is sensitive and rapld and not as llmlted by the quallty of DNA as the RFLP method. The focus of this article Is the use of the PCR for typing genetlc markers, and we will address specllcally the special considerations that arise from applylng DNA ampilflcatkm and typing technology to forensk materials.

INTRODUCTION In 1984, a 3-year-old girl disappeared from her family’s home in the desert. A year and a half later, the top of a small skull was discovered within 2 miles of the missing child’s Corresponding author and present address: Cetus Corporation, 1400 53rd St, Emeryville, CA 94608.

residence. Both the girl’s family and the authorities want to know if this is the little girl’s skull. In another incident, a woman was brutally raped and killed. Three men admitted being involved in events leading up to the crime, and one of them confessed to the rape and murder. However, his confession was not entirely consistent with some of the physical evidence from the crime scene. Is it possible to sort out the role each of these men played in this heinous crime? In particular, is it possible to identify the source of the biological fluids and hairs left at the scene? Five years ago, these questions would have been difficult or impossible to answer. These cases would have remained unsolved or ambiguous had it not been for the advent of technology that enables the genetic typing of minute amounts of DNA. This technology is based on an enzymatic process called the polymerase chain reaction (PCR) that generates millions of copies of a specific sequence of DNA. Since the conception ( I , 2) and original applications (3-5) of the polymerase chain reaction by Kary Mullis and members of the Human Genetics group at the Cetus Corporation, this DNA amplification technology has penetrated all areas of molecular and cellular biology at an unprecedented rate. The PCR has been used successfully in applications ranging from medical diagnostics, allowing early detection of a variety of diseases and infections, to very basic research, enabling scientists to bypass laborious cloning steps and to generate and study mutations at sites previously unmutable by other methods. The PCR and some of its applications have been reviewed in this journal (6) and elsewhere (7-13). PCR-based technology is one of two approaches to DNA analysis currently being applied in forensic science. It has been used in casework since 1986, although the system was not widely available until early 1990. The other method involves the detection of restriction fragment length polymorphisms (RFLPs). RFLP analysis currently is the dominant DNA technology in forensic analysis. The RFLP method was adopted by the FBI for forensic application over a year ago and was in use by two private companies in the United States prior to 1988. However, many observers believe the method eventually will be superseded by the PCR-based approach. The advantages and disadvantages of the two approaches will be discussed in a later section.

THE POLYMERASE CHAIN REACTION The polymerase chain reaction is an enzymatic process by which a specific region of DNA is replicated over and over

0003-2700/9110383-0002$02.50/0 0 1990 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 63,NO. 1, JANUARY 1, 1991

again to yield several million copies of a particular sequence. The boundaries of the region to be amplified are specified by oligonucleotide primers complementary to the 3' ends of the sequence of interest. The PCR occurs in steps characterized by different temperatures. First, the doublestranded template DNA is denatured by heat. The single strands are then cooled in the presence of a great excess of sequence-specific primers; these primers anneal to complementary sequences on the target DNA. Finally, the primers are extended in a template-directed manner by a DNA polymerase to yield new double-stranded DNA products containing the sequence of interest. This step marks the end of a cycle, and the process begins again with the heat denaturation of the DNA strands, which are now present in twice the amount relative to the beginning of the previous cycle (Figure 1). Extension products of the size defined by the primers rapidly accumulate because each strand present a t the end of a cycle serves as a template in the next cycle. In theory, after 30 cycles of amplification, the PCR should yield lo9 copies of the sequence of interest from each template added at the start of the reaction. In practice, the yield is on the order of 10 million copies (17). This level of amplification is sufficient to allow the DNA from individual cells to be analyzed (17, 18), a truly remarkable feat. When the PCR was first conceived, the Klenow fragment of E . coli DNA polymerase was employed for the primer extension step. Because this enzyme is thermolabile, it had to be added at the beginning of each extension step. The most significant advance in PCR methodology was the substitution of DNA polymerase from the hot springs organism Thermus aquaticus for the Klenow enzyme (17). The T. aquaticus enzyme (Tuq),coming from a heat-stable organism (191, is itself stable to the repeated cycles of high temperature required for the PCR. This feature of the enzyme greatly simplified the automation of the temperature cycling since this enzyme needs to be added only once a t the beginning of the reaction, rather than at the start of each new cycle. In addition, Tu9 polymerase affords a much greater degree of specificity and higher yield to the polymerase chain reaction (17);regions up to 10 kbases in length can be amplified, although amplification efficiency declines with increasing length of the target sequence (20).

GENETIC MARKER TYPING Background. The genetic uniqueness of individuals (identical sibs excepted) is a central tenet of human biology. This uniqueness is defined by the combination of genetic markers that an individual inherits from his or her parents. Genetic markers can be analyzed at the level of protein variation or a t the level of DNA sequence variation. Proteins are encoded by genes on chromosomes, and the human genome carries two copies of every chromosome, one copy derived from each parent. Genes are located at a particular position (locus) on the chromosome, but they may exist in alternate forms, termed alleles, which differ a t the DNA sequence level. Different alleles may give rise to proteins that exhibit different properties, or there may be no detectable differences. An individual possessing two identical copies of a particular gene, or copies that give rise to a single protein type, is said to be homozygous a t that locus. An individual carrying different alleles a t a particular locus is said to be heterozygous. The goal of genetic marker typing is to determine which alleles are present at genetically variable loci. In the field of forensic science, the focus of biological evidence analysis traditionally has been directed toward the detection of genetic variation expressed a t the level of blood group and soluble protein markers. These serological typing methods involve the electrophoretic separation of proteins and/or the immunological identification of blood groups from body fluids.

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Figure 1. Graphic representation of the first three cycles of the polymerase chain reaction. The template DNA in the polymerase chain reaction can be any piece of double-stranded DNA containing the sequence of interest ranging in size from the PCR product itself to a human chromosome. The denaturation step typically is performed at 94 O C . The temperature is lowered to an empirically determined temperature between 37 and 72 O C to allow the primers (arrows)to anneal to complementary sequences in the template, and the temperature is raised to 72 OC to allow Taq DNA polymerase to extend the primers. The wavy lines indicate newly synthesized DNA; note that the primer is incorporated into this new strand and defines the 5' end of the product. The straight lines labeled 3' or 5' indicate strands that served as templates in the previous cycle. Note that products longer than the desired PCR product are generated from the original template in this example. These products, defined at only one end by a prlmer, will accumulate at a rate of 2n, where n equals the cycle number. The desired amplification product is accumulating at a rate of 2" - 2 n , and the 2n term becomes negligible after about 10 cycles (only 1 out of 50 fragments are longer than the desired product). Since most PCR protocols specify over 25 cycles ( 74- 76), the larger products will not interfere with the analysis of PCR products. Genetic variation in proteins can easily be detected by these methods because some changes in a protein's amino acid sequence will affect the overall charge of the protein, and hence its electrophoretic mobility, while other changes can alter an antigenic determinant and affect protein-antibody interactions. The advent of DNA typing technologies has generated considerable excitement in the forensic community. One clear advantage of genetic marker typing a t the DNA level is the potential to detect genetic variation beyond that which results in a detectable change in a physical property of a protein. In fact, the human genome is rich in polymorphic sequences that lie outside of amino acid coding regions (21). These sequence variations between individuals can be exploited, ultimately

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ANALYTICAL CHEMISTRY, VOL. 63, NO. 1, JANUARY 1, 1991

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Flgure 2. Analysis of PCR products generated from primers flanking the region of polymorphism. In this example, the polymorphism is a single base pair change in the EcoRI recognition sequence (*). The primers are complementary to conserved sequences on either side of the polymorphism, as indicated by the arrows. Two types of PCR products are generated from this mixed template: one (A) containing the EcoRI recognition sequence and the other (B) containing the altered sequence. Three methods used to analyze the products are illustrated. By use of the dot blot method, the PCR products are fixed to a nylon membrane. The two labeled sequence-specific oligonucleotide (SSO)probes are hybridized to separate identical strips of the membrane under conditions that allow only perfectly matched sequences to remain bound to the products on the membrane. Probe 1 will hybridize specifically to PCR products containing sequence A, and probe 2 will hybridize to products containing sequence B. Consequently, heterozygous products (a mixture of sequence A and sequence B products) will hybridize to both probes while homozygous products will hybridize to only one probe. Restriction enzyme digestion followed by gel electrophoresis allows facile determination of genotype when a recognition sequence is affectedby the polymorphism. Judicious choice of the flanking primer positions allows unambiguous patterns for homozygous and heterozygous types to be obtained. Enzymatic DNA sequence analysis of PCR products provides the actual sequence of the product through the polymorphic region. The polymorphic position is marked by the asterisk (*). Sequences are read from the bottom to the top of the gel. The sequence of the type A product is 5’-GAATTC whereas the type B product is 5‘-GAACTC, with the complementary sequences being 5’-GAATTC and 5’-GAGTTC, respectively. The heterozygous sequence is distinguished from the two homozygous sequences by the presence of both a T and a C residue at the polymorphic position.

to the point of absolute identification. Additional advantages specific to the analysis of forensic samples will be discussed in the next section. Use of the PCR for Genetic Marker Typing. The polymerase chain reaction can be applied to the analysis of genetic variation in two general ways. First, polymorphic regions can be amplified and the sequence variation detected in a separate step. Second, allelic products can be amplified selectively. These two strategies will be discussed in turn. In the first approach, primers are positioned in a region of constant sequence on either side of the sequence polymorphism; the amplification products contain the polymorphic region in the middle of the fragment at a known distance from the ends (Figure 2). The products can then be analyzed by a variety of methods. Three methods of analysis currently being used or slated for future use on forensic samples are illustrated in Figure 2.

(1) Hybridization with Sequence-Specific Oligonucleotide Probes. In a dot blot assay, PCR amplification products are fixed to a nylon membrane and incubated with labeled sequence-specific oligonucleotide (SSO) probes. The labeled SSO probes will hybridize only to products containing the specific sequence and will give rise to a dot upon visualization of the label (Figure 2). The dot blot approach has been modified such that the probes are immobilized on membrane strips and incubated with labeled PCR products (22). This modified assay forms the basis of the forensic DNA analysis kit developed a t the Cetus Corp. ( 2 ) Electrophoretic Analysis of PCR Products. PCR products that differ in size due to repeat sequences, deletions, or insertions can be analyzed directly by gel electrophoresis. Electrophoretic analysis of PCR products containing a polymorphism that affects a restriction enzyme recognition sequence is illustrated in Figure 2. PCR products containing

ANALYTICAL CHEMISTRY, VOL. 63, NO. 1, JANUARY 1, 1991

the recognition sequence will be cleaved by the restriction enzyme to form smaller fragments that are easily distinguished on a gel. Heterozygous samples contain both digested and undigested products. PCR products that do not differ in size but contain sequence mismatches can be distinguished by denaturing gradient gel electrophoresis (23, 24). (3)DNA Sequence Analysis. The ultimate analysis of PCR products is to determine the actual DNA sequence of the amplification product (Figure 2). Direct enzymatic sequencing of PCR products is possible if the products are partially purified or if amplification is carried out such that one strand is produced in excess over the other strand (23,25).However, the nature of some polymorphisms (deletions, insertions, repeated sequences) would make the DNA sequence of PCR products generated from heterozygous templates extremely difficult to interpret; an additional step, such as denaturing gradient gel electrophoresis (23, 24), must be included to separate the allelic products prior to the sequencing step. A number of additional ways to analyze polymorphic PCR products have been reviewed ( 6 ) ,but it is not clear what roles, if any, those methods will play in the forensic field. The second strategy for using the PCR to analyze genetic variation is to amplify alleles selectivelyrather than to amplify all possible alleles in one step and then to determine in a separate step the alleles actually present. Allele-specific amplification is achieved by taking advantage of the fact that some primers with a mismatch to the template sequence at its 3’ end cannot be extended in the PCR (26-28). A set of primers can be designed such that the 3’ nucleotide of the primer aligns with a particular polymorphic base in the target sequence, one primer for each allele. If the 3’ base of the primer is complementary to the target sequence, amplification will occur. Allele-specific amplification is achieved if the primer containing the complementary 3’ base is the only primer extended on each allele. Whether or not the 3’ mismatched primers can be extended must be determined empirically for each set of primers and alleles. A recently developed fluorescent dye system provides a dramatic, colorful method to follow allele-specific amplification (29;diagrammed in Figure 3). Methods for the automated analysis of allele-specific amplification products without a gel electrophoresis step have been proposed (29). This fluorescent label method is applied most simply to two-allele genetic markers, although modifications can be made to analyze additional alleles. In addition, this method can be combined with other analytical methods to allow differential typing of a number of genetic markers amplified together in a single reaction. Use of the RFLP Typing Method. As mentioned in the Introduction, PCR-based systems are not the only DNA typing systems being applied to forensic casework. The other system detects restriction fragment length polymorphisms (RFLPs). Briefly, the process involves digestion of human genomic DNA to completion with a restriction enzyme, separation of the restricted fragments on an agarose gel, transfer of the fragments to a nylon membrane, and incubation of the membrane with a radioactive probe. The probe hybridizes to complementary sequences in the DNA fragments attached to the membrane, and the bound probe is visualized by autoradiography. The fragments to which the probe hybridizes vary in size between individuals. The sizes of these fragments are determined and compared between the evidence and reference samples. There are two kinds of genetic variation that can lead to different size restriction fragments. First, if there is a mutation in the recognition sequence of the restriction enzyme being used, then smaller or larger fragments can be generated

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Figure 3. Use of fluorescent-labeled allele-specific primers to type genetic markers. Alleles 1 and 2 differ at a single position as Indicated. Primers are synthesized with either an A or a C at the 3’ end to allow specific binding to allele 1 or allele 2, respectively (A). Red (R’) and green (G’) fluorescent labels attached to the 5’ ends of the allelespecific oiigonucleotide (ASO) primers allow red PCR products to be generated from the allele 1 sequence and green PCR products to be generated from the allele 2 sequence. In this example, the PCR products from both alleles are identical in size and will migrate together during gel electrophoresis. Uniquely colored products are obtained for the 3 genotypes upon excitation of the dyes (B). Samples containing only one allele (from a homozygous individual) will yield either a red or a green band. Samples containing both alleles (from a heterozygous individual) will yield both red and green PCR products that will occupy the same position in a gel. The combination of the red and green dyes yields a yellow band.

(Figure 4A). Second, DNA sequence elements such as insertions, deletions, or repeated units can increase or decrease the distance between restriction sites (Figure 4B). It is this second type of genetic variation that is being exploited for forensic purposes. There is a highly polymorphic class of sequences in the human genome containing a variable number of tandem repeats (VNTRs). A commonly used set of VNTR probes was developed by Alec Jeffreys (30);they detect length polymorphisms a t many loci. These probes yield a fairly complex pattern of bands on the autoradiogram and can be difficult to interpret. Another type of VNTR probe is specific for polymorphism at a single locus and yields only one or two bands on the autoradiogram (31). A series of these single locus probes is being used by the FBI and other laboratories for casework.

FORENSIC ASPECTS OF GENETIC MARKER ANALYSIS Biological fluids are key elements in the investigation of many kinds of crimes. In sexual assault cases, the assailant(s) may leave semen in the victim, on clothing, or at the location of the assault. In homicide cases, the victim’s blood may be found on the suspect’s clothing or on a weapon in the suspect’s possession. The goal of the forensic serologist is to determine, by genetic marker typing, whether the biological evidence could have originated from a particular individual, either victim or suspect. The serologist decides what typing tests

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Table I. Criteria for Useful Forensic Genetic Markers

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U Figure 4. Two types of genetic variation revealed by RFLP analysis. (A) When a mutation occurs in the recognition sequence of the restriction enzyme chosen for the analysis or creates a recognition sequence, different size products will be produced and detected. (B) The length of DNA between two given restriction sites can vary due to insertions, deletions, or, in this example, repeated units. Depending on the size and the difference in number of the repeated unit between the restriction sites, the resulting fragment length differences can be detected and measured.

should be done based on the source, age, and condition of the materials, choosing those tests that will provide the greatest discrimination. If a single genetic type obtained from the evidentiary sample does not match the reference type of the individual in question, then the individual can be excluded as the donor of the biological fluid with absolute certainty. If, on the other hand, the genetic types of the individual are concordant with all of those obtained from the evidence, then common origin is possible. A genetic typing match, however, does not constitute an identification; some proportion of the general population may also share the same array of genetic marker types. The larger the excluded population, the greater the weight of the evidence. The serologist presents the typing test results along with the appropriate statistical interpretation to the jury or judge who then weighs this evidence with the other circumstances of the crime to render a verdict. Selection of S u i t a b l e F o r e n s i c Genetic M a r k e r s . Individuals can be distinguished by their genetic marker profile. The more markers compared, the more likely differences between individuals will be revealed. However, not all genetic markers are suitable for forensic applications, and they must be carefully selected. The criteria by which genetic markers are evaluated for forensic use are outlined in Table I. There are some factors that relate to the method of analysis and others that relate to the nature of the marker. The dual goals driving the selection of forensic genetic markers are to enhance discrimination power and to extend the range of evidence samples from which useful information can be obtained. It is worth underscoring the importance of having available an analytical method that requires only a small amount of

material for typing forensic samples. Frequently the amount of evidentiary material available for analysis is quite small and irreplaceable. The less of a sample that must be consumed in an analysis, the greater the opportunities for retyping the sample and analyzing additional markers. In addition, it is desirable to retain a portion of the sample for subsequent testing by the opposing side; this practice is part of the check and balance system for science that enters the adversarial judicial system. The three criteria relating to the nature of a useful genetic marker are based on the information that can be obtained from genetic studies and the statistical analysis of gene (allele) frequencies. An index by which genetic markers can be judged is the capacity of the marker to distinguish between individuals. If the genotype frequencies of the marker within a defined population are known, then the probability that two individuals chosen at random from this population will possess the same genotype (PI)can be calculated (32). If the population is in Hardy-Weinberg equilibrium for this marker, then the expected genotype frequencies can be calculated from the observed allele frequencies. The index value, known as the discrimination power (PD), is equal to 1 - PI and is the probability that two random individuals will possess different genetic types for the marker being tested. Higher PD values indicate a greater individualization potential because a larger portion of the population can be excluded. For a single marker system, the greater the number of alleles (degree of polymorphism) and the more even their distribution throughout a population (higher the heterozygosity), the higher the power of discrimination. The PDfor combinations of marker systems is equal to 1 - (product of PIvalues for each marker system); as one adds more marker values, the cumulative PDvalue approaches unity. Markers must be inherited independently (present on separate chromosomes or in linkage equilibrium if on the same chromosome) for this PD to be valid. Genotype frequencies for a particular marker can vary from population to population. Therefore, it is important to collect allele frequency data from relevant reproductive populations so the appropriate statistical evaluation of the typing results can be made. H i s t o r i c a l Perspective. The best known and most venerable of the genetic markers used in forensic testing is the AB0 blood group system; the A B 0 markers are quite stable and can be detected in most types of biological evidence. The other blood group markers (e.g., Rh and MN) are rarely used in forensic testing because they exhibit variable stability in bloodstains and are not found in semen, saliva, or other biological evidence. During the 19709, the forensic genetic typing tools were expanded to include the electrophoretic detection of protein markers. Of the many protein markers found in blood, approximately 10 proved to be sufficiently robust to be typed in bloodstains. Unfortunately, only a few protein markers are present in other kinds of biological evidence such as semen. While the range of samples that could be typed was not greatly extended by the addition of protein marker typing, the a priori probability of a chance match was reduced to less than 1 in 200 for relatively good quality bloodstains and to 1 in 10 for

ANALYTICAL CHEMISTRY, VOL. 63, NO. 1, JANUARY 1, 1991

semen evidence. However, evidence samples that have undergone significant environmental deterioration will not yield comparable results. Forensic biology entered the DNA era in the mid-1980s. Three independent research groups demonstrated that DNA could be extracted from virtually all forms of biological evidence (33-35). Of particular note, it was found that the DNA from sperm could be differentially extracted from the DNA of other cell types (33). This extraction method greatly benefits the typing of sexual assault evidence which typically contains a mixture of sperm from the assailant and epithelial cells from the victim. Genetic marker types of the assailant and the victim can be distinguished from each other, a distinction not possible with the blood group and protein markers. The initial efforts within the forensic community to develop DNA typing focused on restriction fragment length polymorphism (RFLP) analysis. The VNTR sequences detected by the commonly used single locus probes are assigned to a bin, and an allele frequency is determined for each bin. These frequencies are combined for the set of probes used to give a very high power of discrimination. Closely following the introduction of RFLP analysis for typing forensic samples was the introduction of a PCR-based typing system suitable for the analysis of forensic samples. Advantages of PCR-Based DNA Typing of Forensic Samples. PCR DNA amplification technology is simple, rapid, and unparalleled in sensitivity and applicability. As such, DNA typing using the PCR has distinct advantages over the RFLP method. First, PCR-based typing is easier and quicker to perform and does not involve the use of radioactivity. PCR typing results can be obtained in 1-2 days compared with weeks for RFLP results. PCR-based typing also is amenable to complete automation, which can reduce operator error and increase the number of samples that can be typed in a given period of time. A second advantage of PCR-based typing is its sensitivity; as little as 1 ng of DNA or less is required for analysis. Consequently, DNA extracted from single hairs (36,37),small blood, semen, and 'saliva stains (20, 38, 39), organ tissue (S. Swarner, unpublished results), and even fragments of bone and teeth (E. Blake and S. Swarner, unpublished results) has been typed by using the PCR. PCR-based DNA typing systems also are particularly well-suited for the analysis of the small samples usually collected in sexual assault cases. Moreover, because PCR-based typing methods consume so little DNA, analysis of the samples can be repeated, a great asset for forensic work. In contrast, RFLP analysis is limited to samples yielding between 50 and 500 ng of DNA, and repeat analysis is not always possible. Another advantage of PCR-based typing systems is that they are less sensitive to the degree of DNA degradation than the RFLP-based systems. Progressive degradation of DNA samples leads to loss of RFLP bands, making analysis of type impossible (K. Konzak, unpublished results). In contrast, degraded DNA samples extracted from ancient tissues (40-42) and unpreserved 10-15-year-old case materials (E. Blake, unpublished results) have been amplified successfully. Amplification of degraded DNA samples requires only that the average fragment size in the sample be equal to or exceed the size of the region(s) to be amplified (R. Reynolds, unpublished results). The simplicity and sensitivity of the PCR coupled with the ability to type degraded DNA samples greatly expands the numbers and types of cases that can be analyzed. To illustrate this point, data from 44 cases were compiled. These cases originally were submitted for RFLP analysis and were later sent to one of us (E. B.) for PCR analysis. Of these 44 cases,

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RFLP results had been obtained for 9 of them. Unambiguous PCR results were obtained for 40 (91%) of the cases. Samples from the four cases for which PCR results were not obtained also did not yield RFLP. One of the failed cases involved a urine sample; two of the cases involved samples that did not contain adequate material for PCR analysis but were attempted nonetheless. The fourth case yielding no PCR results contained DNA extracted by the company that performed the RFLP analysis. Clearly, many samples not amenable to RFLP analysis can be successfully typed by using PCR-based technology. PCR-based typing falls short of RFLP-based methods in one area-power of discrimination. However, as new marker systems are developed for typing by PCR-based methods, this gap will be closed.

THE HLA DQa TYPING SYSTEM Currently there is one PCR-based genetic marker system commercially available and validated for forensic casework. The Cetus Corp. has developed and released a PCR-based kit for determining the HLA DQa type of DNA extracted from forensic materials. This system has been used on over 200 cases to date. The two cases described in the Introduction came from this group; their resolution will be described in a later section. The genetic system on which the kit is based was originally developed to facilitate research on immune system diseases and histocompatibility testing ( 4 , 23, 43, 44). It takes advantage of the high degree of polymorphism found in the major histocompatibility gene complex (MHC) proteins. Specifically, a polymorphic coding region of the gene for the a subunit of the DQ protein is amplified by using flanking primers situated in regions of constant sequence. Based on extensive sequence data of this polymorphic region, eight DQa alleles have been defined (23,44,45;T. Bugawan and H. A. Erlich, unpublished data). The alleles are divided into four major types (Al, A2, A3, and A4) with A1 and A4 further divided into subtypes (Al.1, A1.2, A1.3, A4.1, A4.2, and A4.3) (21). The current typing system (22) is designed to differentiate six of the eight alleles (Al.1, A1.2, A1.3, A2, and A3; A4.1, A4.2, and A4.3 are grouped as A4). Twenty-one genotypes are defined by these six alleles. Unlike the simple examples illustrated earlier in which the polymorphism consisted of only a single base pair change, the DQa sequence variants differ from each other at as many as 27 positions on a 239 base pair fragment. The detection assay of the typing system in the kit is a modified dot blot procedure. The standard dot blot format entails fixing denatured PCR products on a series of nylon membrane strips, one strip for each probe, as illustrated in Figure 2A. With the modified procedure, termed the reverse dot blot format, the single-stranded sequence-specific oligonucleotide (SSO) probes are affixed to the nylon membrane strips and the PCR products remain in solution for hybridization to the immobilized probes (22). An advantage of immobilizing the probes rather than the PCR product is that one hybridization can be done simultaneously to all probes. The DQa PCR primers are biotinylated at their 5' ends, enabling binding of a streptavidin-horseradish peroxidase complex to the hybridized PCR products. The peroxidase enzyme catalyzes the conversion of a colorless substrate to a colored precipitate (Figure 5 ) . The sequence-specific binding of the biotinylated PCR products to the immobilized SSO probes and the peroxidase reaction are occurring on the surface of the membrane, the result of which is a blue dot at a defined position on the strip. There are nine positions occupied by different probes on the test strip. The four major types (Al, A2, A3, and A4) are distinguished by four probes; however, these probes do not differentiate the A1 subtypes or the A4 subtypes. The Al.1,

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EXTRACTION OF DNA FROM FORENSIC SAMPLES

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2*4

Figure 6. Immobilizedprobe test strips for typing DQa. The reverse dot blot patterns for three heterozygous DQa genotypes are illustrated. Each of the 21 genotypes has a unique pattern of dots. The roles of the “all” and “all but 1.3” probes in the typing system are described in the text. The “all” probe dot has a hatched pattern to indicate its lower intensity relative to the sequence-specific probe dots.

A1.2, and A1.3 subtypes are differentiated by four additional probes. One probe is specific for the Al.l allele, and one is specific for the A1.3 allele. The third probe is complementary to a sequence common to the A1.2, A1.3, and A4 types, and the fourth probe can hybridize all DQa types except A1.3. This probe is required to distinguish between the 1.2,1.3 and 1.3,1.3 genotypes; all other genotypes can be distinguished without it. A positive control probe is included on the strip to which products of all DQa alleles can bind. This probe is applied at a reduced concentration relative to all other probes such that it should normally give the least signal of any probe. This probe serves as an indicator of adequate amplification of the DQa alleles in the test sample and of possible procedural errors such as incorrect hybridization conditions. An example of three heterozygous DQa types is shown in Figure 6. The relative intensities of the “all” probe and the typing and subtyping dots are compared to make a typing call. The 21 DQa genotypes are determined by their unique patterns of blue dots on the test strips. Over 1400 individuals from 11populations have been typed for D Q c y (46) by using the typing system described above. The observed DQa genotype frequencies do not differ significantly from the expected frequencies based on the assumption of Hardy-Weinberg equilibrium (46). The DQa marker system by itself has a f D of 0.94 in the Caucasian population (46). The f D values for the individual traditional protein markers range from less than 0.1 to about 0.7 (47). Clearly the DQa system alone provides a much higher discrimination potential than any individual traditional system. A significant effort is being made to develop additional marker systems that can be analyzed by PCR-based methods to combine with the DQa

c PCR AMPLIFICATION (denature, anneal, extend)

c REVERSE DOT BLOT TYPING

Flgure 7. Four physical steps in the analysis of genetic markers by a PCR-based method. The DNA extraction step is the most variable in the analysis because of the tremendous variety of materials obtained as evidentiary samples. Extreme care must be taken not to introduce exogenous DNA into the reaction tube during the reaction preparation step. The PCR amplification and typing steps are easy to perform since they are automated and repetitive, respectively, but they are sensitiwe to temperature changes and must be carefully monitored. Once the typing step is completed, the results must be interpreted.

marker to increase the overall PD values. Some of these systems will be discussed in the last section. POTENTIAL PROBLEMS AND CONCERNS Prior to the incorporation of any new tool into forensic practice, the tool must be thoroughly and critically evaluated for possible sources of error. Every analytical method can fail due to operator carelessness, inappropriate application, or poor reagents. Fortunately, these types of problems usually signal themselves, warning the operator that the test should be repeated. In the case of genetic typing analysis, the more important concern is to identify factors that have the potential to cause one genetic type to look like another. Consequently, much research effort is being devoted to identifying factors that have the potential to cause one genetic type to appear as another type. In addition, understanding the factors affecting the yield, efficiency, and reliability of the PCR is critical for increasing the range of application to evidentiary samples. The analysis of genetic markers using a PCR-based typing system can be divided into four operational steps: DNA extraction, reaction preparation, marker amplification, and marker typing (Figure 7). Once the samples are typed, the results must be analyzed. The particular circumstances of each case greatly affect the interpretation of typing results, and they must be taken into account. The potential problems that may arise a t each step of the analysis will be discussed below in addition to the special concerns arising from the nature and condition of forensic samples. DNA Extraction and Reaction Preparation Steps. The quality of the DNA sample is the most critical factor in the PCR in that it determines the success of the amplification and typing steps. The forensic scientist is challenged with extracting DNA from stains on all types of fabrics and surfaces, hairs, swabs, bits of tissue, and pieces of bone. Methods have been developed and are evolving for the extraction of DNA from these materials. The sensitivity of the PCR has raised concerns that exogenous DNA introduced into samples might be amplified, leading to potential mistyping or ambiguous results. There are four sources of exogenous DNA to be aware of. First, the samples may become contaminated upon collection from the crime scene. However, at the recommended sensitivity level of the DQa typing test, this source of contamination appears negligible (P. Fish and K. Comey, unpublished results). Second, forensic samples may arrive at the crime laboratory

ANALYTICAL CHEMISTRY, VOL. 63, NO. 1, JANUARY 1, 1991

already containing a mixture of fluids from more than one person. Mixed samples are common with sexual assault evidence and cannot be avoided. However, methods to separate sperm cells from epithelial cells have been developed to allow the sperm DNA to be typed separately from the female’s DNA. In contrast, mixed blood and/or saliva samples cannot be sorted, and the possibility of a mixed sample must be considered when interpreting typing results. The third source of exogenous DNA is another genomic DNA sample extracted in the laboratory. In some cases, there is no way to obtain a fresh sample of the same material. This potential source of contamination and limitation also is sometimes faced by the clinician when PCR is used to analyze biopsy or prenatal, chorionic villi samples. The fourth source is PCR products carried over from previous amplification reactions. Carryover PCR product is an important contaminant to consider and protect against because of the large number of PCR product molecules generated during amplification. Once an extracted test sample is contaminated by either genomic DNA or PCR product, there is no way to “decontaminate” it. However, if the sample is contaminated with PCR product of a particular marker, other marker loci could still be amplified and typed. Positive steps can and should be taken to control contamination of the test sample. The first step is to separate the work areas for the four steps of the typing system. The DNA extraction and reaction preparation steps can be performed in the same room but preferably in separate areas. The amplification and typing steps ideally should be performed in a separate room with supplies that never leave the room (Figure 8). In addition, separate sets of pipettors should be dedicated for extraction, for reaction preparation, and for handling amplified DNA products in the second room. Ideally, a set of positive displacement pipettors should be dedicated for addition of extracted DNA samples to the PCR tubes during the reaction preparation step. Use of positive displacement pipettors eliminates the possibility of contaminating the barrel of the pipettor and transferring DNA to another reaction. All pipeting should be performed with care to prevent the formation of aerosols and small drops. A small drop of volume 0.1 p L from a completed amplification reaction could easily contain loQcopies of PCR product. Considering that a 100-ng sample of genomic DNA contains only 3 X lo4 copies of a single-copy gene, the introduction of a PCR product-containing drop to that sample could lead to a false positive or mistyping result since the PCR product is itself an extremely efficient template for the reaction. Although the extraction of all DNA samples is done in one area, it is important to separate and extract samples according to the relative amounts of DNA they are likely to contain. For example, a liquid blood reference sample can contain over 100 pg of DNA while a vaginal swab or a single hair may contain only nanogram quantities of DNA. It would not be prudent to isolate DNA from a suspect’s or victim’s liquid blood sample a t the same time as the evidentiary samples, which usually contain less DNA. Other steps for preventing contamination and mistyping of samples have been reviewed and discussed elsewhere (48, 49). Judicious choice of positive and negative controls will signal a contamination problem, and the source can then be traced and eliminated. Contamination of a sample is revealed most frequently by the presence of more than two alleles. In many cases, there will be sufficient DNA to repreat the amplification and typing, provided the template DNA has not been contaminated. The final consideration regarding the DNA extraction step is that some of the materials on which bodily fluids are deposited release agents upon extraction that inhibit Taq polymerase. Certain components in blue demin and some

9

ROOM 1 AREA 1

I

EXAMINATION AND SEPARATION

OF EVIDENCE MATERIALS

1 AREA 3

AREA 2 EXTRACTION OF EVIDENCE MATERIALS

PREPARATION

ROOM 2 AREA 1

AREA 2

STORAGE

Figure 8. Ideal laboratory setup for the analysis of genetic markers in forensic materials using the PCR. Rooms 1 and 2 should be separated by a closed door, and no item or equipment exposed to amplified products should be brought into room 1. A clean area in room 1 should be available for logging in and examining evidence, and a

bench near a microscope should be dedicated for the extraction of

DNA from evidentiary samples. A biological hood equipped with a UV

germicidal lamp is useful for preparing PCR reactions. Storage of pipettors and racks in this hood under the UV lamp are recommended to damage any errant DNA and render it unsuitable for amplification (discussed in text). Room 2 should contain space for the thermocycler and a dedicated water bath, a gel electrophoresis area for the evaluation of the amplification reactions, and an area for typing the products. In addition, this room should contain a refrigerator or freezer for the storage of PCR products. Not all laboratories can be organized into two separate rooms. If pre- and post-PCR analysis steps are confined to a single room, then extreme caution must be taken, and dedicated work areas and equipment must be established and maintained. leathers are particularly effective inhibitors of the PCR (E. Blake, unpublished results). In addition, a heme-containing component released upon extraction of shed older blood stains also has proved inhibitory; this inhibition is not observed with relatively fresh bloodstains on the same types of materials (S. Walsh and R. Higuchi, unpublished results). We expect to gather more information about PCR inhibitors as the technique is applied to more cases. Methods to alleviate inhibition of the PCR are being investigated; frequently the inhibition can be overcome simply by diluting the DNA sample prior to addition to the reaction or by adding more Taq polymerase or primers to the amplification reaction (E. Blake and S. Walsh, unpublished results). A new extraction method for forknsic samples using Chelex appears to reduce inhibition of amplification (S. Walsh, manuscript in preparation). Amplification a n d Typing Steps. Obtaining the correct DNA marker type of a sample when the PCR is used is the end result of a series of complex biochemical processes that are sensitive to a variety of factors. A critical factor for the amplification cycle is temperature. Specifically, strand separation during the denaturation step of the amplification cycle can be affected by small changes in temperature. If there are melting temperature differences between the alleles, then reduction of the denaturation temperature could result in differential denaturation of some alleles. PCR primers cannot anneal to strands that have not separated; consequently, alleles on these strands will not be amplified. This phenomenon was originally observed with the DQa system with a 1.1,4 sample of denatured genomic DNA that occasionally typed as 4,4 (K.

10

ANALYTICAL CHEMISTRY, VOL. 63, NO. 1, JANUARY 1, 1991

Comey, unpublished results). The role of denaturation temperature in this phenomenon was subsequently identified and studied systematically (R. Higuchi, and S. Walsh, manuscript in preparation). A t measured denaturation temperatures of 88-89 "C, the 4 allele is amplified but the 1allele is not when denatured genomic DNA is used. At 87 OC, neither allele is amplified, and a t temperatures between 90 and 96 "C, both alleles are amplified. When native duplex genomic DNA is used, there is no amplification of either allele a t 88-89 "C; preferential amplification due to differential denaturation has not yet been observed with nondenatured genomic DNA. However, this phenomenon also has been observed with denatured heterozygous type 1,3 DNA (K. Comey, published results) and theoretically could occur with heterozygous type 1,2 DNA as well because of the higher GC base content of each of the 1alleles (1.1,1.2,1.3), relative to the other major alleles. Differential amplification of VNTR loci also has been observed and attributed to differential denaturation due to differences in GC base content (20). This potentially serious problem can be completely avoided by comparing the actual and set temperatures of the thermocycler and making any necessary adjustments. In addition, tubes must be pushed down into the wells of the thermocycler so the entire surface of the tube is in contact with the metal block (R. Higuchi, personal communication). A control DNA sample with one of the 1alleles and the 2,3, or 4 allele may be run with every amplification series, also. As new marker systems are developed, particularly GC-rich VNTR loci, their potential for differential denaturation should be evaluated. Specific annealing of an oligonucleotide primer to its complementary target sequence also is sensitive to temperature variation. Too high a primel-template annealing temperature will preclude amplification whereas too low a temperature will allow amplification of nonspecific products, thus reducing the yield of specific products. Nonspecific products can be a problem if the typing process involves the direct analysis of the size of amplified products; they are much less of a problem when specific hybridization with a probe is used in the typing process. Again, the potential problem can be eliminated by using a calibrated thermocycler and the recommended conditions for the optimal amplification of DQa or other markers. Typing steps involving the hybridization of PCR products and probes frequently have a small temperature window in which reliable results can be obtained. For example, the D Q a typing system has a 2-deg window; temperatures above and below the window can affect hybridization. At temperatures above the window, some signals become weaker, and at temperatures below the window, cross-hydridization of the subtyping probes occ~rs(V. Phillips and s. Walsh, unpublished observations). Careful monitoring of the temperature during the hybridization step eliminates this potential problem. Inadequate denaturation of the PCR products prior to adding them to the membrane strips could result in a low level or even a lack of binding to the probes. Theoretically, there may be conditions under which one allele may not denature to single strands while other alleles are fully denatured, giving an aberrant type. Again, attention to the recommended times and temperatues will prevent these kinds of potential mistyping results. The polymerase chain reaction is sensitive to the concentrations of the reaction components. In particular, concentrations of M F above or below the optimum can dramatically reduce the specificity of the reaction (12). Presumably, if kit reagents are used, then Mg2+,primer, Taq DNA polymerase, and deoxyribonucleoside triphosphate concentrations will be controlled. Special Concerns Associated with Forensic Evidence Samples. Degradation and Damage. DNA extracted from

A.

M

0' 2' 7' 15'30'90' M

202 137 94

56

12

B. 0'

2'

7'

15'

30'

90'

LABCABCABCABCABCABCO

)c 242 bp

Figure 9. Amplification of DNA degraded with DNaseI. (A) Human genomic DNA (0.1 mg1mL) was digested with DNaseI (1 nglpL) for varying amounts of time. Samples of 800 ng of digested DNA from the timepoints indicated were run on a 1% agarose gel. The gel was stained with ethidium bromide to visualize DNA. Lanes marked "M" contain X DNA digested with EcoRI and HindIII. (B) Three amounts of DNA (200, 50, and 20 ng) from each timepoint were added to separate FCR mixes containing primers to amplify the 2391242 base pair region of DQa. An equal volume from each completed amplification reaction was run on a 3% NuSieve/l % agarose gel. Lanes marked A contained 200 ng of DNA in the PCR, B contained 50 ng, and C contained 20 ng. Lanes marked "L" contain a 123 base pair molecular weight siting ladder.

forensic samples frequently is degraded to some degree and has been exposed to environmental conditions of varying degrees of harshness. There is considerable interest in understanding what effects, if any, these various forms of insult have on the PCR and typing. DNA degradation can be simulated in the laboratory by exposing human genomic DNA, isolated from whole blood samples, to a variety of degradative enzymes. Degradation of DNA with DNase I results in cleavage of the DNA a t random positions with the average size of the fragments decreasing with increasing time of digestion (Figure 9A). These DNA fragments were used in amplification reactions with primer pairs defining different sizes of D Q a products to look at the effect of random degradation on the PCR. As expected, PCR products can be generated from degraded DNA provided the average size of the fragments is a t least as long as the desired product. Amplification of the 242 base pair DQa region is shown (Figure 9B). The PCR products generated

ANALYTICAL CHEMISTRY, VOL. 63, NO. 1, JANUARY 1, 1991

1

11

Table 11. Proportion of PCR Product Strands Containing an Amplification Error

,*

I I

I I *

I

I

I

Flgure 10. Possible “jumping”pathway on damaged DNA template.

In this hypothetical example of PCR jumping, there are no templates containing an intact target sequence and many of the templates contain damage sites through which Taq polymerase cannot extend (*). Primers 1 and 2 are extended until Taq polymerase encounters a damage site or the end of a fragment. In the next cycle, this incompletely extended product serves as a long primer on the same or another template. Again it is extended to a damage site or end of a fragment. This process continues until a full-length product is generated. This full-length product will become double stranded in the next cycle and will be amplified exponentially in succeeding cycles. If the original DNA sample Is heterogeneous or contains a mixture of types, then the final PCR products may contain a composite or mosaic sequence from two or more alleles.

from these degraded DNA fragments typed correctly for DQa, indicating that degradation per se has no effect on the typing accuracy of this system. An interesting observation made during these and other degradation studies in our laboratory is the increased yield of specific PCR product from slightly degraded DNA relative to “high molecular weight” DNA (DNA appearing to be greater than 21 kb in length in test gels). Presumably, the slightly degraded DNA denatures more easily and binds more primers in the first cycles of the PCR, thus increasing the effective number of starting template molecules. In addition to exposure to degradative enzymes, forensic samples frequently are exposed to UV radiation via sunlight. UV radiation induces formation of covalent dimers between adjacent pyrimidine bases; it also can induce covalent linking between bases on opposite strands and cross-linking of DNA to protein. Experimental studies show that UV irradiation of purified genomic DNA results in reduced amplification by the PCR; above a saturating dose, no amplification occurs. The Tuq polymerase appears to stop at sites of pyrimidine dimer formation rather than generate products that will be amplified (50). These findings provide an explanation for the recent suggestion that PCR mixes be irradiated just prior to addition of template DNA to reduce significantly the possibility of contamination by PCR product carryover (51); irradiated contaminants will not be amplified by Tuq polymerase, and PCR products will be made only from the added template DNA. Irradiation of simulated forensic evidentiary samples (liquid and dried samples of blood and semen) had little effect on the PCR. The protein present in these samples appears to have a protective effect with regard to UV damage generation. However, irradiation did reduce DNA recovery from dried semen stains (50);the DNA from these stains appears to be part of a large protein-DNA complex. Neither enzymatic degradation nor UV irradiation of DNA samples resulted in a false positive or mistyping result in these studies. However, it has been proposed that, with badly degraded DNA, a composite or mosaic sequence may result from incompletely extended products “jumping” from one template to another (17, 52, 53) (Figure 10). With highly degraded DNA, mosaic sequences or “shuffle clones” (17) can

cycle no. of error substitution

initial copies of DNA

1

2

5

r

1 10 100

0.25 0.025 0.0025

0.125 0.0125 0.00125

0.015 0.0015 0.00015

1/ 2?+’ 0.1/2’++’ 0.01/2‘++’

result from a primer that has been partially extended on one allelic template, annealing in a subsequent cycle to the other allelic template. This can occur rarely on undegraded DNA as well (17) but does not affect the results of oligonucleotide probe typing (H. Erlich, unpublished results). Since it takes several cycles to generate a full-length double-stranded molecule from fragments of DNA, we do not expect this phenomenon to present a major problem provided there is some intact target sequence present. Preliminary experiments indicate this to be the case. Issues of Fidelity. Mutations such as base substitutions, insertions, and deletions in the DNA sequence could create new alleles or, in some cases, switch among known alleles at a particular locus. Concern has been voiced that sequence errors generated during amplification by Tuq polymerase could result in the conversion of one genetic type to another, leading to a false positive result or no result. The nucleotide misincorporation rate of Tuq polymerase ranges from about 2 nucleotides for every lo4nucleotides incorporated to l nucleotide for every lo5 incorporated, depending on the amplification conditions (17, 54, 55). T o generate a false result, the misincorporated nucleotide would have to occur at a site that is critical for typing. For example, if a SSO dot blot or restriction site polymorphism system is used to type the marker, then the misincorporated nucleotide must be located at a site critical for hybridization to the probe or within the restriction enzyme recognition sequence, respectively. In addition, the various methods used to type genetic markers all analyze a collection of molecules, not single molecules. Since it is common to type a sample containing millions of PCR products, the specific error would have to be present in a significant number of the products to be detected above the background of error-free products. Given the above reasoning, it is clear the cycle number a t which the error occurs and the number of starting template molecules play a large role in determining the risk of mistyping as a result of nucleotide misincorporation. The mathematical demonstration of the effects of cycle number and starting template molecules, assuming a misincorporation rate of 1out of lo4,is shown in Table I1 (from ref 12). If only a single copy of DNA was subjected to amplification and an error occurred somewhere in the region defined by the primer pair during the first cycle, then one-fourth of the products will contain the misincorporated nucleotide at the end of the amplification. If the error occurred during the fifth cycle from a single copy, less than 2% of the products will have the incorrect sequence. This worst case scenario based on a single starting template is not realistic, however, because special amplification conditions are required to detect a single template. To minimize this and other potential problems, PCR amplification conditions that require greater than 10 initial copies to generate a detectable product are recommended for forensic analyses. In addition, a more typical forensic template minimum is 100 copies (350 pg of genomic DNA), and 2 ng is recommended by the Cetus Corp. for DQa typing. With this higher number of starting template molecules, even if there was an error introduced during the first cycle, only one-quarter of 1 % of the products would contain the error. It is important to emphasize this analysis for an error occurring at any position

12

ANALYTICAL CHEMISTRY, VOL. 63, NO. 1, JANUARY 1, 1991 !

I

'

'

I

3

Xther

1 2 . 2 STD

"'

] ]

3,3 STD

-

- . -

4,4 STD

A

Figure 12. Wa types of bone from skull and of missing gM's parents. PCR products generated from standard DNA samples (left) and reference blood samples from the missing girl's parents and DNA extracted from the skull (right) were fixed in duplicate on a nylon membrane for conventional dot blot analysis. An explanation of the types is described in the text.

Flgure 11. Photograph of skull cap found in the desert from which scrapings and a small section were taken for DNA analysis.

in the PCR product; the probability that the error will be in a position that can lead to a mistyping is quite low. Nucleotide misincorporation could occur on damaged templates at depurinated sites. Although this type of damaged molecule will likely introduce an error during the first cycle, it is unlikely all of the molecules will be damaged at the same position, much less at a critical position. Consequently, using the above reasoning, we do not expect this type of damage to contribute to mistyping results. EXAMPLES OF APPLICATIONS OF THE PCR TO FORENSIC CASEWORK Many cases go unsolved because the ability to obtain genetic information from the available biological evidence is somehow compromised. As discussed earlier, not all protein markers are expressed in all biological fluids, and typing of those expressed in a particular fluid or tissue may be limited by the age of the sample. PCR-based DNA analysis generally is not limited by sample origin or age. These features of the PCRbased technology, in addition to its sensitivity, frequently allow for the analysis of forensic specimens that are not amenable to protein or RFLP typing. The two case examples noted in the Introduction illustrate the power and utility of PCR-based DNA typing. The missing person case involves the disappearance of a 3-year-old girl from her parents' home in the desert in October 1984. Her body was never found, and she was listed as a missing person. In March of 1986, a portion of a small skull was discovered within 2 miles of the parents' residence. Anthropological examination of the skull cap revealed it to be of human origin, from a child between the ages of 2 and 5 years. This conclusion certainly is consistent with it being from the missing 3 year old, but without fresh tissue, teeth, or the rest of the skeleton, no positive identification could be made. In July of 1988, the skull cap was sent to one of us (E.B.) for DNA analysis. If DNA typing were possible, it could then be determined whether the skull was genetically compatible with a child of the missing girl's parents. Material for DNA analysis was obtained by scraping the external and internal surfaces of the skull and by chipping off a piece of bone (Figure 11). The skull scrapings and bone chip were digested in the presence of sodium dodecyl sulfate, dithiothreitol, and proteinase K, extracted with phenol/chloroform, and concentrated to obtain a DNA sample for amplication. DQa typing was performed by using the traditional dot blot procedure by fixing the PCR products, in duplicate, to a nylon membrane and hybridizing DQa sequence-specific probes to the membranes. The mother and father of the missing child were determined to be DQa types 3,4 and 4,4, respectively (Figure 12). A child

Table 111. DQa Types of Reference Samples from a Rape/Homicide Victim, Her Boyfriend, and Three Suspects

donor

DQa type

victim boyfriend suspect 1 suspect 2 suspect 3

1.2, 4 1.1, 1.1 1.1, 1.2 2, 3 1.1, 1.1

of these two people must have either a DQa type 3,4 or 4,4; the two types are equally probable. Were the skull unrelated to these parents, then any type would be possible. DQa types 3,4 and 4,4 occur in about 19% of the Caucasian population, so there is an 81% chance of excluding an unrelated individual. The results of amplification and typing of the skull samples are shown in Figure 11. Amplification of the scrapings did not produce a typable product; the scrapings therefore serve as a control for surface contamination on the skull bone. In contrast, the bone chip material did amplify, and it typed as DQa 3,4. This typing is consistent with the skull cap coming from the missing child, but it does not allow an absolute identification. The DNA from the bone chip recently was amplified for mitochondrial sequence analysis (56). A mother shares identical mitochondrial sequences with her children; the father's mitochondrial sequence is not passed to his children. The mitochondrial sequence analysis of the bone and the child's mother revealed that they share identical sequences through two highly polymorphic regions. I t has been estimated that less than 1%of the Caucasian population would have this particular mitochondrial DNA sequence (56). The second case involving a rape/homicide provides an illustration of the impact PCR-based DNA typing can make on the ability to analyze sexual assault cases. In this case, three men were involved in the crime, but the role of the three assailants was in question and could not be determined by conventional protein marker or blood group typing. The three suspects were connected to the victim through the fraudulent use of her credit cards prior to her death. On the night of her death, the three men were drinking together and presumably went to the victim's home together. One of the three men confessed to the rape and murder (suspect l), a second was held on the basis of a bloody footprint a t the scene (suspect 2), and the third was linked by association with the other two suspects (suspect 3). An unusually large number of hairs were left at the scene of the crime, and these were sent along with semen stains and reference samples from the victim, her boyfriend, and the three suspects for DNA analysis. The objective of the analysis was to determine if any of the suspects could be eliminated as donors of the evidence specimens. The DQa types of the reference samples are listed

ANALYTICAL CHEMISTRY, VOL. 63,NO. 1, JANUARY 1, 1991

Reference wa:4

Evidence wa:4 3 2 1

3 2 1

II

::I

3

3 3.3SM. 3 4.4SM.

1

11 I *

0

/a. e

3 ]

.3 0

e

1,l std.

0

’

0

13

3

1.4 victim

suspect 1 1.1

boyfriend 1.1

suspect 3

1

1.1

I

su-2

I

1.2 all 1.3 but 2 3 4 all 1.1 4 1.3 1.3 - .

1.1,l.l

I

suspect 3

I

suspect 2

12,3

2.3

7

I

*

i

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1.1,1.2

suspect 1

1.1,l.l

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I NA

blank

Flgure 13. Conventkmal and reverse dot bbt analysis of DNA samples extracted from the rapdhomicide case samples. (A, left two) PCR products generated from both reference and evklence samples were fixed to four positions on a nylon membrane in duplicate, as indicated. The membrane was cut into four strips, and each strip was hybridized with one of four labeled SSO probes, similar to the example in Figure 2A. The reference samples include PCR products generated from standard (std) DNA samples of known type (std) and reference blood samples from the victim, her boyfriend, and the three suspects. Note that these probes do not distinguish the three DQa 1 subtypes. The subtypes of the reference samples were determined in a separate analysis of the samples by using the reverse dot blot procedure (see 8). The evidence samples include 10 hair samples, 2 of which did not amplify and 1 of which showed a mixed type. The typing results of the epithelial cell (e. cell) fraction and the sperm fraction obtained from a semen stain also are shown. There appears to be some sperm DNA remaining in the epithelial cell fraction, leading to a mixed type. The sperm fraction types unambiguously as DQa 2,3. (8, right) Reverse dot blot strips were used to determine the 1 allele subtypes of the suspects and the victim’s boyfriend. The strips and the method of detection are described in the text.

in Table 111. The victim and the three suspects are distinguishable from one another, but the victim’s boyfriend and suspect 3 have the same type. The origin of any evidence with a 1.1,l.l type cannot be determined from this information alone. Visual examination of the hairs revealed that many of them appeared to originate from an individual or individuals of African ancestry, consistent with the suspects’race (the victim was Caucasian). A number of the hairs were cut a t one end, indicating the donor(s) had recently had a hair cut;these hairs were not subjected to PCR analysis since they did not contain a hair root. For the initial analysis, 18 hairs distinguishable from the victim were selected; 3 of these were contaminated with blood. DNA was isolated from the hairs and amplified for D Q a typing. Thirteen hairs typed as DQa 2,3, and three hairs gave no result. The remaining two hairs gave a mixed type of 1.2,2,3,4 (Figure 13). This result is consistent with a mixture of the victim’s type with a type 2,3, as well as several other combinations. Additional hairs, including some that could have come from the victim, were subsequently typed. From this set, eight had the victim’s type (1.2,4), three were determined to be DQa 2,3, and two hairs had the mixed type seen with the other set of hairs tested. Sperm-containing semen samples were differentially extracted from a vaginal swab and a stain on a sheet. Intact sperm cells were separated from epithelial cell debris in the sample prior to extraction. The two fractions were analyzed independently to allow DNA from the sperm to be typed without interference from the female component of the sample. The sperm DNA from both the swab and the stain samples typed as DQa 2,3, consistent with the type of the hair donor (s).

Table IV. DQa Typing Results Compiled from 125 Cases typing results

no. of cases

7O of cases

inclusion exclusion inconclusive

72 30 23

58 24 18

Table V. DQa Typing Results of Individual Samples from 125 Cases no. of samples

no. typed clearly

7O typed

sample single hair sperm evidence bloodstain

183 233 86

69 166 23

38 71 27

clearly

The man who confessed to the rape and murder (suspect 1)can be eliminated as the donor of the hairs and semen found a t the scene; his type is DQa 1.1J.2. Suspect 3 and the victim’s boyfriend also are eliminated as donors of these specimens. The man who matched the bloody footprint at the scene (suspect 2) had a DQa 2,3 type and accordingly cannot be eliminated as the source of the hair and semen. The DQa 2,3 type occurs in only 3% of the Black population. The jury for this case convicted this suspect of rape and murder. The man who confessed to the rape and murder was convicted as a co-conspirator, a lesser charge. Data from 125 forensic cases that could not be resolved by either conventional or RFLP analysis have been compiled to illustrate the utility of PCR-based DQa typing specifically. Of these 125 cases, analysis of 82% yielded conclusive results.

14

ANALYTICAL CHEMISTRY, VOL. 63,

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In 58% of the cases, the suspect was included by the results and in 24%, the suspect was excluded as the donor of the sample(s) (Table IV). In Table V, the actual samples analyzed from the cases are grouped and the percent of each group that gave an unambiguous DQa type is listed. The apparently low 27% value for evidence bloodstains is due to inhibition of amplification. As methods to reduce inhibition have been developed, the number of blood stains that can be amplified and typed has increased to over 40% (E. Blake, unpublished results).

FUTURE DIRECTIONS Automation. Automation of the PCR was instrumental in its proliferation through all areas of biological science. The job facing organizations that want to develop databases of DNA types would be greatly facilitated if the DNA extraction and typing steps also were automated. The reverse dot blot system is highly amenable to automation or a robotics system since it requires essentially only the addition and replacement of a series of buffers at different temperatures. If DNA sequencing becomes a typing system of choice, then the use of automated sequencing procedures may be desirable. However, the system must be able to read heterogeneous sequences derived from heterozygous individuals. New Markers. As described in previous sections, the ultimate success of DNA typing in the forensic field by a PCR-based method lies in the development of additional marker systems. The markers must meet the criteria outlined in Table I, and it would be desirable if newly developed markers were not associated with any disease states, to minimize fears of intrusion of privacy. Many of the traditional protein markers used for forensic typing are suitable by these criteria for development as DNA typing markers, but the DNA sequence of the protein is not available for most of these candidates. One protein marker that has been developed into a DNA typing system is group specific component, Gc (57). A region of the Gc gene contains two polymorphic restriction enzyme sites that can be amplified on the same PCR product. The amplified DNA is digested with the restriction enzymes and analyzed by gel electrophoresis as described in Figure 2. The three common Gc alleles give rise to six possible Gc genotypes, each of which yields a unique restriction pattern (57). A reverse dot blot assay for Gc, to be used in conjunction with the DQa marker assay, is being developed in collaboration with the Cetus Corp.; this system will include four other two and three allele markers. Polymorphic mitochondrial sequences and VNTRs, which are exploited by the RFLP method, are being developed into PCR-based typing systems. There are two advantages of using mitochondrial sequences. First, there are more copies of the particular sequence of interest per cell than with single copy nuclear encoded sequences, which increases the chances for successful typing. Second, the mitochondrial sequences are not heterozygous, since only the maternal mitochondrial DNA is passed to the child. This feature makes DNA typing by sequencing very plausible, particularly in an automated format. A dot blot format for typing mitochondrial sequences has been developed at the Cetus Corp. (56). The value of this system in routine forensic typing has not been determined, but it clearly is useful in the investigation of missing persons since a mother and all her children share the sequence. VNTR elements provide another source of polymorphic sequences, and several VNTRs have been amplified by the PCR (20,5843). Since VNTRs are characterized by fragment length differences, the amplification products can be analyzed directly by gel electrophoresis. These pieces of DNA should not share the problem of band shifting due to modified DNA seen with the RFLP method because they are simply products of the PCR, not pieces of forensic DNA. However, several

potential problems of using the PCR to amplify repeated sequences can be anticipated. First, if the DNA sample is degraded, it is possible that the larger of the two alleles would not amplify, leading to an apparent homozygous type for a heterozygous sample. This effect has been observed with the MCT118 amplification system (R. Reynolds, unpublished results). The second potential problem with amplification of VNTRs is that products with fewer repeat units may be preferentially amplified over longer products because Tuq polymerase is more efficient on shorter fragments (20). This problem could introduce ambiguity into the typing result since one or more signals will appear less intense than the other(s). In general, a significantly weaker signal is attributed to an additional participant in the crime or to contamination. In those samples containing a long and a short VNTR sequence, the potential exists that the long VNTR signal would go undetected or be misinterpreted. However, at least one VNTR system does not appear prone to this problem for the alleles identified to data (62, 63). The third potential problem that may lead to ambiguous results is the potential for Tuq polymerase to “slip” while extending the primer through a repeated region. Under these conditions, some regions of the template will be copied into the product more than once, yielding an array of products longer than the target sequence. Slippage can be minimized by using VNTRs with longer repeat units. Jeffreys et al. (20) also have observed “out-of-register annealing”,resulting in the production of PCR products containing the wrong number of repeat units, when too many cycles or too much DNA is used to amplify VNTR regions. These problems are not trivial and must be thoroughly investigated on forensic samples before applying the system to active casework samples. Education and Training. Another area that is becoming increasingly important is education and training. The successful application of this incredibly sensitive technology requires that the people performing the work be highly trained and aware of potential problems and their solutions. Future efforts to develop on-going training workshops for criminalists using the PCR will go a long way to ensuring and maintaining the high-quality work the forensic field demands.

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RECEIVEDfor review June 11, 1990. Accepted October 11, 1990. Portions of the work described in this review were supported by grants from the National Institute of Justice (Grant 86-IJ-CX-0044) and the State of Caliiornia Department of Justice. Points of view or opinions expressed in this document are those of the authors and do not necessarily represent the official position or policies of either agency.