SPECIAL REPORT
DNA Profiling New tool links evidence to suspects with high certainty
John I. Thornton, University of California, Berkeley
In 1983, immigration authorities refused to allow a boy to enter the U.K. because they were skeptical that he was the natural offspring of a Ghanian woman who was a legal U.K. resident. With DNA fingerprinting, Alec J. Jeffreys, a geneticist at the University of Leicester, was able to show that there was only one chance in tens of billions that the boy was not the woman's son. After reflecting on the fact that the world's population is only about 4 billion, the British government conceded the birthright of the child. This was the first time DNA typing was used in a forensic setting. 18
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Since then, DNA analysis of biological evidence has been successfully introduced in over 100 American criminal cases and in about 50 in Britain. Thousands of disputes about paternity also have been settled with this technique. This year, the number of forensic laboratories in the U.S. that undertake DNA analysis, commonly known as DNA profiling or DNA fingerprinting, has grown markedly, and the FBI (Federal Bureau of Investigation) began widespread use of the technique in December 1988, after a year of practical tests. In crimes of violence, blood is often shed. In sexual assault, seminal fluid may be released. Biological fluids are most often found as dried stains or, in the case of sexual assault, obtained as vaginal swabs that have been dried. The various genetic markers in these fluids form the basis for the scientific analysis of biological evidence. These markers are blood group antigens, polymorphic proteins (proteins that are electrophoretically separable into several forms because of amino acid variations arising from DNA mutations), and DNA polymorphisms (different forms of DNA arising from sequence differences in the particular gene or sequence of DNA material). When biological evidence is presented in court, a connection is made between the crime and the suspect by comparing the genetic markers found at the scene of the crime with those of the suspect. Making sense out of the results requires population statistics describing the frequency of each of the marker types. These frequencies are essentially probabilities and, therefore, can be multiplied together to determine the probability that an individual possesses a specific profile of markers. The significance of DNA profiling is the high degree of certainty with which it can link a suspect to a particular piece of evidence. Conventional blood typing using blood group antigens can rule out suspects whose blood types differ from that found in the evidence. The information provided by conventional blood typing tests is, therefore, only exclusionary. That is, the forensic serologist can make a definitive statement as to the origin of a stain only when the genetic markers found in the stain differ from those found in the suspect or victim. If the stain contains the same red blood cell antigens as the suspect, the analyst is limited to stating only that the suspect may have been responsible for the stain. Because individuals with
each blood type comprise a fairly large fraction of the population, this kind of test does not allow the evidence to be linked to the suspect with a high degree of probability. The use of polymorphic proteins in blood stains or semen can establish a relationship between the evidence and a suspect with a higher degree of certainty. With tests of this type, the typical probabilities that a specific piece of evidence is not from a particular suspect range from 1 in 100 to 1 in 10,000, depending on the particular proteins and the number of proteins that are tested for. Though the statistics are more meaningful, definitive statements involving polymorphic proteins are still only exclusionary in nature. In contrast, the use of DNA polymorphisms can result in probabilities as low as 1 in 1019 that two matching samples of biological evidence are not from the same individual. In other words, the chance that identical DNA patterns exist between two randomly selected individuals can be vanishingly low, and the link between biological evidence and a particular suspect based on DNA polymorphisms can be extremely strong. For this reason, DNA typing is likely to revolutionize the analysis of biological evidence. Because violent crime is such a pervasive element of U.S. society, any technique that links suspects positively with crimes or rules them out with a great deal of certainty is important. Homicide is the leading cause of death of Americans aged 15 to 23 and is the fourth leading cause of death in all Americans under the age of 65. Further, a woman born in the U.S. today has about a 12% chance of being forcibly raped in her lifetime, and in some urban areas it is nearly 60%. Unless the homicide rate declines appreciably, 2 million U.S. citizens who are now alive will eventually be murdered. DNA analysis didn't spring full-formed like Minerva out of the head of Zeus. Rather, it is the result of an evolutionary process. To put a discussion of DNA profiling in perspective, it is useful to consider the usual analytical schemes that are applied to the examination of biological evidence. When a reddish brown stain is found at a crime scene, forensic chemists immediately ask several questions. First: "Is this stain blood?" Historically, many different approaches dating back to the 6th century in China have been used to answer this question. The modern approach, however, is based on the composition of blood, and most tests now take advantage of the reactivity of the major protein of red blood cells, hemoglobin. The heme group in hemoglobin promotes the oxidation of a colorless (leuco) base, such as tetramethylbenzidine, to a colored complex. This characteristic is used as a presumptive test for blood. These tests, which consist of applying a leuco base to a possible blood stain, are simple, quick, and very sensitive; some tests, such as the test using luminol as the base, can detect blood in dilutions as low as 1 in 105 to 107. Once it has been determined that a stain is probably
blood, the species must be identified. Generally, the question is whether the stain is human blood, but occasionally, such as in crimes involving endangered animal species or poaching, it is necessary to identify a nonhuman stain. In either situation, the analysis is based on the ability of an antibody to recognize serum proteins in a species-specific manner. The origins of this type of analysis can be traced back to the late 1800s, when the nature of immunological reactions was beginning to be elucidated. By the early 1900s, these methods were being used in a forensic context. The modern techniques of species identification are
Forensic bloodstain analysis follows a logical scheme Presumed bloodstain
t Is it blood? (peroxidase reactive)
X
X
Yes
No
\ Is it human? (species-specific antibody) No
ABO typing (Lattes, absorptionelution, ELISA)
Yes
Polymorphic protein tests (gel electrophoresis)
DNA typing (RFLR ASO/PCR)
Each stain that is presumed to be blood is subjected to a logical series of tests. First, blood is tested for by applying a colorless (leuco) base to the stain. In bloodstains, the heme group in hemoglobin promotes the oxidation of the leuco base to a colored complex. If the stain is blood, it is tested to see if it is human blood, using antisera that differentiate human albumin from albumin of other species. If the stain is human blood, it is next subjected to one or more of three major different types of tests—ABO blood group typing, tests for polymorphic proteins, or DNA typing. The methods used depend on the nature of the question to be answered, the amount of stain available, the length of time since the blood was shed, and personal preference of the examiner. Three major kinds of ABO typing are available: the Lattes test, the absorption-elution test, and enzyme-linked immunosorbent assay (ELISA). The Lattes test is used infrequently because it can be employed on only relatively new stains. Gel electrophoresis is used to study up to 15 different polymorphic proteins found in bloodstains. The techniques used to test for DNA differences are restriction fragment length polymorphism (RFLP) analysis and allele-specific oligonucleotide (ASO)/ polymerase chain reaction (PCR) DNA amplification.
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Special Report usually based on Immunoelectrophoresis or precipita tion reactions involving antigen-antibody complexes. Again, these tests are relatively quick and simple. They are based biologically on species evolution. As species have diverged from one another, species-specific mutations have occurred that result in amino acid substitutions in the proteins. It is these mutations that distinguish human albumin from, for example, chim panzee or chicken albumin. Appropriate antisera can differentiate albumin or globulins of one species from those of another. The next set of questions in the typical bloodstain analysis concerns specific genetic markers found in blood. The markers tested for can be either red blood cell surface antigens, serum protein antigens, or poly morphic proteins from red blood cells or serum. The particular genetic markers that are chosen depend on the amount of stain available, the length of time since the blood was shed, and, to some extent, personal preference and laboratory facilities available to the examiner.
Blood typing In 1900, Karl Landsteiner, an Austrian physician and immunochemist, described the first human ge netic polymorphism, the blood group system that classifies blood into types A, B, and O. These are examples of serological markers; that is, they are based on immunochemical reactivity. The ABO antigens are glycolipids and glycoproteins attached to the cell mem brane of the erythrocyte (red blood cell); the lipid or protein part of the antigen anchors it to the blood cell's membrane and the carbohydrate portion acts as the antigenic determinant. Landsteiner's discovery en abled investigators to connect blood samples to vic tims or suspects. Three principal methods of ABO typing are avail able. The Lattes method tests for antibodies that rec ognize blood type A or Β antigens. A person's ABO type determines the specificity of antibodies in the serum; a type A person has anti-B antibodies, a type Β has anti-A, and a type Ο has both. The Lattes test exploits the presence of these antibodies to identify blood type. If the antibodies are present, appropriate washed red blood cells agglutinate when added to the crust of a blood stain. The Lattes method must be used only on relatively new stains because antibody pro teins tend to be unstable. Typically their reactivity falls off rapidly in the first month and is unlikely to persist for a year. Given this constraint, the absorption-elution test is generally viewed as a better choice for ABO typing. For this test, the ABO antigens must remain immuno logically reactive, so that added antibodies can bind to the stain. The antibodies can subsequently be eluted when exposed to elevated temperatures (about 60 °C). These eluted antibodies then agglutinate the appro priate type of washed red blood cells. The third technique, enzyme-linked immunosorbent assay (ELISA), is somewhat newer and less commonly used. It is an immunological method for detecting antibodies or antigens in biological materials, based 20
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Tests for blood utilize the oxidation promoting activity of the heme group in hemoglobin
Reduced tetramethylbenzidine (colorless)
Oxidized tetramethylbenzidin (colored)
To test for blood, a colorless (leuco) base, such as tetra methylbenzidine, is applied to the stain in the presence of an oxidizing agent, such as hydrogen peroxide. If the stain is blood the heme group in the hemoglobin promotes the oxidation of the leuco base to a colored complex. Some tests, such as one using luminol as the base, can detect blood in dilutions as low as 1 in 10 5 to10 7 .
on the assumption that when either antibodies or antigens are coupled to enzymes, the resulting complexes will retain both immunological and enzy matic activity. By varying the design of the assay, either antigens or antibodies can be detected. ELISA shows great promise for increased sensitivity as well as for automation. The British Home Office Central Research Establishment has done much basic research, as well as system automation, in this area. In addition to the ABO red blood cell antigens, some 50 other polymorphic antigens have at least the potential of being exploited for forensic purposes. Other constituents on the red blood cell membrane, such as the Rhesus (Rh) factor, have from time to time been used in forensic analyses, but they have proven to be somewhat unreliable and are therefore used much less frequently than the ABO types.
Protein polymorphisms In the 1950s and 1960s, advances in protein chemis try allowed possible genetic polymorphisms in pro teins to be investigated. The technique that paved the way was gel electrophoresis, which is based on the fact that proteins are polymers of neutral and posi tively or negatively charged amino acids. Electrophoretic methods are so sensitive that a difference be tween proteins of one charged amino acid may result in a mobility difference on the gel. The commonly studied proteins were those that were readily available in sufficient quantity from blood cells and serum. Population geneticists soon realized that the ability to distinguish polymorphic proteins by electrophoresis could be an extremely useful tool for investigating possible genetic differences. Proteins from different species are immunochemically differ ent, and these differences arise from amino acid varia tions resulting from DNA mutations. Thus, because some amino acid substitutions result in different net protein charges, the proteins display a range of mobilities on the electrophoretic gel. This made more powerful evolutionary studies possible. In addition, multiple forms of particular proteins exist within a single species. And some enzymes have several electrophoretically separable forms that re-
fleet differences in DNA, expressed as p r o t e i n polymorphisms. By studying protein polymorphisms, geneticists were indirectly investigating DNA differences, since pro teins are coded by DNA sequences. A necessary crite rion is that these polymorphisms follow the laws of genetic inheritance so they are meaningful genetic markers—that is, the genetic marker must be precise ly determined by the parents rather than some ran dom or whimsical biochemical expression. This is nec essary for establishing population statistics. As the number of known polymorphisms increased, it be came apparent that studying the differences in pro tein polymorphisms would be a particularly useful method of establishing biochemical individuality. For forensic purposes, it then became a question of wheth er the proteins in a dried bloodstain were stable enough to be analyzed electrophoretically. Currently, crime laboratories can test for up to 15 different proteins that are stable in bloodstains. Test ing is conducted more or less routinely for phosphoglucomutase (PGM), esterase D (EsD), erythrocyte acid phosphatase (ΕΑΡ), adenylate kinase (AK), adenosine d e a m i n a s e (ADA), carbonic a n h y d r a s e (CA II), glyoxalase I (GLOI), haptoglobin (Hp), and group spe cific component (Gc).
Carbohydrate fragments of red cell A, B, and Ο antigens determine blood type
Sexual assault evidence The analytical approach to sexual assault evidence closely parallels that for bloodstains. The most useful information, in the case of rape, is generally found on a vaginal swab taken from the victim during medical examination following the assault. If semen is present on the swab, the source of the semen must be deter mined. The semen is assumed to have originated from the assailant unless the victim engaged in sexual in tercourse shortly before the assault. Unlike blood, the number of useful markers in se men is not particularly great. Generally, tests are run for only two or three genetic markers. Historically, the first of these was the ABO blood type. It was shown in the 1920s that about 80% of the population secrete into their proteinaceous body fluids the same ABO oligosaccharide antigens that are present on red blood cells. These individuals are known as "secretors." By typing their body fluids, it is possible to determine their ABO types. Clearly, this is important in a sexual assault investigation, since the most common type of evidence are semen and, to a lesser extent, saliva. The other genetic marker used extensively in the investigation of sexual assault is PGM. Genetically inherited differences or polymorphisms in this en zyme can be tested for with electrophoresis. As with the secreted ABO antigens, the seminal PGM type is the same as that expressed in blood. Thus, given the results of a swab analysis, and a blood sample from a suspect, it is possible to draw conclusions regarding the source of the semen. This, of course, can be done only if the PGM type of the victim is determined, as this genetic marker also will be present on the swab and may otherwise produce confounding results. The analyst must then decide, on the basis of the markers
Type Β determinant OH
Type Η determinant Ac = Acetyl Gal = Galactose GalNAc = N-Acetylgalactosamine GlcNAc = A/-Acetylglucosamine Fuc = Fucose
A, B, and 0 blood types are determined, respectively, by the A, B, and Η determinants. (The odd naming of the determinant for type 0 arises for complicated historical reasons.) These antigenic determinants are the carbohy drate portions of the A, B, and 0 antigens. The oligosac charide antigens are attached to either lipid or protein carrier molecules that are anchored in the red cell mem brane. The Η antigen is the precursor for both the A and Β determinants, which differ from each other in only one sugar.
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Special Report present, if the suspect can be included as a possible source of the semen, or whether the genetic markers have excluded him. Typical probabilities for exclusion based on PGM polymorphisms are between 50 and 75%. Again, definitive statements can be made only in an exclusionary sense.
The sequence of bases in one strand of DNA determines the sequence of the other
DNA profiling With DNA typing, however, a sample of biological evidence can be linked much more positively with a suspect. The essential components of DNA are two sugar-phosphate backbones (one for each strand of the double helix) and the four nitrogenous bases— adenine (A), guanine (G), cytosine (C), and thymine (T). These bases extend into the interior of the helix as stairs in a spiral staircase. Each stair is composed of two bases (one from each strand) that specifically rec ognize each other by hydrogen bonds. Every adenine is hydrogen bonded to a thymine on the opposite strand; similarly, each guanine is bonded to a cyto sine. This specific relationship between the two strands is known as "complementary base pairing" and is essential to the biological role of DNA as well as to DNA typing. To put the total amount of information carried by DNA into perspective, the genetic material of humans (the so-called genome) consists of 3 billion nucleo tides. These 3 billion units comprise the 23 pairs of chromosomes, which are continuous strands of DNA ranging in length from 50 million to 500 million nucleotides. Encoded in this DNA is the information for about 100,000 genes. Additionally, about 95% of the DNA is considered "junk"—that is, it may have some as yet unknown function but does not code for any particular gene. In terms of DNA profiling, however, both genes and the noncoding regions are relevant. Given this incredible amount of genetic informa tion, it is not at all surprising that considerable varia tion is found from one person to the next. The knowl edge of this variation is not new, but it was only recently that methods became available to study its extent at the molecular level. The techniques used to study this aspect of biochemical individuality are of two types—restriction fragment length polymorphism (RFLP) analysis, and allele-specific oligonucleotide (ASO)/DNA amplification. The latter is more com monly called PCR (polymerase chain reaction) analy sis. Both techniques are important in forensic chemistry. In 1980, David Botstein, a biochemist at the Massa chusetts Institute of Technology, suggested the RFLP technique of DNA analysis as an approach for map ping the human genome. The method relies on the use of restriction enzymes that specifically cut the chromosomal DNA into shorter strands. These en zymes are isolated from bacteria that use them as a defense mechanism against viral infection. By cleav ing (restricting) the DNA of the invading organism, the bacteria neutralize the viral attack. There are sev eral hundred restriction enzymes available, and each has a specific DNA recognition sequence (composed of approximately four to 12 consecutive base pairs) at which it cuts the double helix. For example, the dou22
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Sugar-phosphate backbone of DNA
Base pairing in DNA DNA is a polymer of a limited number of individual mono mers. The monomers are nucleotides, which consist of a nitrogenous base, a sugar (deoxyribose), and a phosphate group. The nitrogenous bases are the purines adenine (A) and guanine (G) and the pyrimidines thymine (T) and cytosine (C). Nucleotides are linked via phosphodiester bonds to form a backbone of alternating sugars and phosphates (upper structure). The bases protrude off the sides of the backbone and pair with bases of another nucleotide strand through hydrogen bonding (lower struc ture). This process of pairing is not random; A and Τ specifically recognize each other via hydrogen bonds, and G and C do likewise. Therefore, the sequence of one strand determines that of the other through complementa ry base pairing. This concept of complementarity is criti cal to the analytical methods employed in DNA profiling.
ble strand recognition site of the enzyme Eco RI is GAATTC CTTAAG This restriction site, as well as most others, is palindromic—that is, it is the same regardless of which strand is being "read." Wherever this particular se-
Common, straightforward laboratory techniques are used in RFLP analyses Bloodstain
Developed film shows the pattern of DNA fingerprint, which is compared with patterns from known subjects
DNA is extracted from blood cells and .
. . . cleaved into fragments by a restriction enzyme
X-ray film, sandwiched to the membrane, detects radioactive pattern
Replicate patterns, | Pattern from same person another person
quence occurs along the chromosome, the enzyme cleaves the sugar-phosphate backbones in two places in the sequence, resulting in smaller fragments of DNA. The laboratory techniques used in RFLP analysis are fairly straightforward, and commonly employed throughout molecular biology. RFLP analysis starts by isolating DNA from the fluid or tissue of choiceblood, liver, muscle, or semen, for example. The purified DNA is then cleaved with a restriction enzyme to produce smaller fragments. These fragments of DNA are run through an electrophoresis gel to separate them on the basis of size. The smaller fragments move faster in the charged electrical field than the larger ones. When the electrophoretic separation is complete, the fragments of interest are separated into single strands (denatured) with heat. They are then transferred from the gel and immobilized onto a solid nitrocellulose or nylon support with the Southern blot transfer technique, named after its originator, Edward M. Southern, a molecular biologist at Oxford University. Once the fragments are immobilized on the solid support, a radioactively labeled piece of single-stranded DNA, the "probe/" is hybridized to these fragments by means of hydrogen bonding. This is where the concept of complementarity is important. The probe used can either be a specific sequence chosen because it is known to be from a particular gene, or it can be a random fragment of DNA that happens to recognize RFLPs because it has sequences of nucleotides complementary to those of the RFLPs. Both types of probe are in use today. When the radiolabeled support is immersed in a solution containing the probe, those fragments complementary to the probe bind to it. These fragments can then be visualized by means of autoradiography. (An x-ray film is put in direct contact with the probelabeled filter, and bands of exposed film are produced in the positions corresponding to the fragments recognized by the probe.) RFLP analysis is thus possible because of differ-
Fragments are separated into bands by gel electrophoresis
The DNA band pattern in the gel is transferred to a membrane (Southern blotting)
Radioactive | DNA probe binds 1 to specific DNA sequences
Membrane is washed free of excess probe
ences that exist in the sequence of nucleotides that make up the chromosomes. Some of these differences, known as mutations, are important substitutions within a coding sequence of a gene. These may result in a modified protein that is coded for by that particular gene. (An example of this is seen in sickle-cell hemoglobin, in which a single base mutation results in a single amino acid substitution with profound effects on the functional capabilities of the protein). Other substitutions may occur in noncoding regions of the DNA and thus have no effect on any expressed gene product. This variation in the noncoding region can nevertheless be detected by RFLP analysis because the substitution can result in the production or elimination of a restriction enzyme recognition site. This in turn results in different sized fragments produced from the DNA of different individuals. Depending on the combination of enzymes and probes that are used, an RFLP procedure can produce extremely complex patterns consisting of dozens of bands. For example, in 1985, the University of Leicester's Jeffreys developed probes to recognize particular DNA sequences. These probes have proven to be conspicuously applicable to questions of genetic individuality. They result in patterns of more than 40 bands, with the probability of identical patterns between two randomly selected individuals being on the order of 1 in 1019. These multilocus patterns have become known as the "DNA fingerprint" because of their capability to individualize. (In contrast, various mathematical models estimate uniqueness of an ordinary fingerprint as no greater than about 1 in 1010). Although an extreme example, some RFLP systems have been developed that produce patterns of but one or two bands. These single-locus probe patterns have corresponding probabilities of random identity on the order of 1 in 10,000 or so. The probability of the pattern depends on the individual's DNA fragments making up the pattern. Single-locus probes differ from "fingerprint" probes in that the sequence they recognize is found in only one position on the genome, whereas the multilocus probes recognize sequences in November 20, 1989 C&EN
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RFLP analysis splits DNA from different people into fragments of differing length Polymorphic site
Polymorphic site
1000 bases1
One small fragment and one large fragment
Two intermediate size fragments
Because the DNA of different people has different sequences of nucleotides, a particular restriction enzyme cuts their DNA in different places in restriction fragment length polymorphism (RFLP) analysis. For example, in the DNA of one individual, the enzyme (which has a specific DNA recognition sequence) cuts a particular region into one large and one small fragment, say one with 1000 bases and one with 8000. Because the specific recognition sequence occurs at different places in the DNA of another individual, the same restriction enzyme cuts that same region into intermediate-sized fragments, say one with 3000 bases and one with 6000. Fragments can be separated by size using gel electrophoresis and then further analyzed.
many places throughout the genome. Still, if several single-locus probes are used, it is possible to approach the same level of individualization as with multilocus fingerprinting. This approach provides the best features of both systems—pattern simplicity and discrimination power. As might be expected, each kind of probe has its advantages and disadvantages. Multilocus probes result in a definite individualization, but the patterns they produce can be very difficult to interpret. Also, fnultilocus probes require more DNA and the DNA must be in a relatively nondegraded state. The patterns generated by single-locus probes, which require less DNA, are much more easily interpreted but do not provide as much information, and, consequently, not as much discrimination. Both types of probe are currently being used in forensic casework. However, multilocus probes are being used more extensively in cases of questioned paternity. Because analysts can select both probes and restriction enzymes, they have a great deal of flexibility in conducting these types of examinations.
ASO/DNA amplification ASO/DNA amplification combines two procedures— DNA amplification and "dot-blot" analysis. In this form of analysis, the portion of DNA to be identified must be part of a well-characterized gene and the gene of interest must also be polymorphic; that is, 24
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there must exist multiple forms (alleles) of the gene that differ in their nucleotide sequences. The number of different alleles varies among the polymorphic genes, but can be as many as 20. As with RFLP probes, the probes used in ASO analysis are based on the ability to bind DNA sequences according to base pair complementarity. A known sequence for each allele allows allele-specific probes to be produced. Actual analysis for ASOs is somewhat less complicated than RFLP analysis. After the DNA is extracted from the sample, it is denatured (made into single strands) and applied directly to nitrocellulose paper or nylon filters in a process known as dot blotting; no restriction enzyme cleavage or electrophoresis is necessary. The filters are bathed in solutions, each of which contains a different labeled allele-specific probe. Hybridization to the probe in one of the solutions occurs as a result of complementary base pairing between the probe and the DNA. The filters are then washed to remove unbound probe. The results are visualized, generally by autoradiography, and a "genotype" based on the allele-specific probe that binds to the DNA is obtained for that particular gene. DNA may be successfully analyzed by this technique, but combining this technique with an amplification of the DNA represents one of the most ingenious recent developments in molecular biology. The amplification method, PCR, specifically replicates a small region of DNA. It was originally devised in 1985 by Kary Mullis and his colleagues at Cetus Corp., in Emeryville, Calif., and promises to have applications in virtually every biological discipline from evolutionary biology to medical diagnosis. The technique is ideally suited to forensic purposes because it can potentially allow for the DNA profiling of a single human hair, or the most minute bloodstain, a stain that otherwise would be too small to be of value. Also, it allows typing of samples too degraded for RFLP analysis. In principle, if a single copy of the target sequence defined by the oligonucleotide primers is present, amplification is possible. To use PCR, the DNA sequence of bases in the target region must be known. Further, the target region must be polymorphic if it is to be useful in a forensic context. The first requirement is necessary simply for amplification purposes, whereas the second is essential for the development of meaningful population statistics. The genes comprising the HLA (human leucocyte antigen) genetic system fulfill both of these criteria. A specific locus called HLA DQ a, with six typeable alleles, was the first genetic polymorphism to be analyzed by PCR in forensic casework. For PCR amplification, two oligonucleotide primers are used. These primers are sequences of nucleic acid 20 to 30 bases long that are complementary to the DNA on the flanks of the target region to be amplified. They define the ends of the DNA that will be replicated. In the first step of PCR analysis, the DNA to be copied is denatured with heat. The oligonucleotide primers are then annealed to the denatured flanking sequences, one on each strand. Next, the primers are extended along the single-stranded DNA
Lasers make latent fingerprints visible Another recent advance in applying forensic chemistry to the identification of suspects is the use of lasers to develop hidden or latent fingerprints. Some latent fingerprints luminesce when illuminated by the blue green light (4579 and 5145 A) of an argon ion laser. Freshly deposited fingerprints have a greenish yellow fluorescence; older prints tend to luminesce orange. Copper vapor and frequency-doubled neodymium:yttrium aluminum garnet la sers also have been used to develop fingerprints. The first successful use of this technique was the develop ment of a latent fingerprint on a piece of black vinyl electrician's tape, a ma terial from which formerly it was im possible to develop fingerprints.
water. Therefore, the fingerprint con tains only about 10~ 6 g of solids, which are about equally distributed between salts and a witch's brew of organic chemicals, including lipids, polypep tides, amino acids, and vitamins. Be cause fingerprints are very thinly distributed over several square centi meters, the problem is even more challenging.
Latent fingerprints deposited on pa per or other porous substrates have classically been developed with ninhydrin to give a colored complex called Ruhemann's Purple. The prints are then sprayed with a solution of zinc chlo ride to make them fluoresce strongly under laser light. Laser development of most prints may then proceed successfully.
Quite independently of the laser de velopment of fingerprints, it was found that fingerprints can be made visible when they are exposed to the fumes of "Krazy Glue." A reaction between the polypeptides of the fingerprint and the cyanoacrylate of the glue produces a polymer. Though the polymer itself is not luminescent under laser excita tion, it stabilizes latent fingerprints so they can be treated chemically with an efficient laser dye, such as Rhodamine 6G. Many prints that are so faint that they cannot be discerned with the fluorescence from the riboflavin or some other natural fingerprint material that luminesces under laser light be come visible with this technique, some times with spectacular results.
This technique has two major prob lems, however. First, a great many materials exhibit a high level of back ground luminescence that overwhelms the luminescence from a faint finger print. Second, some individuals se crete so little luminescent material in their perspiration that laser develop ment fails to detect their fingerprints. Even the most ideal fingerprints con sist of very little material, typically about 10~ 4 g, of which 98 to 9 9 % is
The problem of strong background luminescence also has been addressed successfully. The time between exci tation and the termination of back ground luminescence, called turn-off time, typically is very short, ordinarily about 1 nanosecond. If a latent finger print is treated with an organic dye that has a substantially longer turn-off time, however, the background lumi nescence can be separated from the fingerprint luminescence. The laser may
templates by the enzyme DNA polymerase, forming double-stranded DNA. After one such three-step cy cle, there are two copies of the target sequence. This three-step cycle is repeated many times, pro ducing copies of the original DNA as well as copies of the primer-defined target DNA. The copies of the copies include only the target sequence, whereas copies of the original DNA extend beyond the target sequence. Because the number of copies of copies grows exponentially, while the num ber of copies of original DNA increases arithmetical ly, the target sequence eventually dominates the reac tion mixture. This cycle of heat denaturation, primer annealing, and enzyme extension is repeated 20 to 30 times, resulting in 2 20 to 2 30 (about 109) copies of the target DNA. Consequently, in a few hours the num
be pulsed to promote luminescence from both the substrate and the finger print. At the end of the pulse, the first nanosecond of decay is ignored, since that luminescence is from the back ground. An imaging gate—an electric circuit that enables only the fluores cence to be imaged and excludes the excitation—then allows the lumines cence decay from the dye to be re corded for a few nanoseconds, after which the process is repeated. For this to work, a dye with a long decay period is needed. Tris(2,2'-bipyridyl)ruthenium(ll) chloride hexahydrate is ideally suitable. The lumines cence lifetime of this compound, when coupled to a cyanoacrylate-fumed la tent fingerprint, is 1.5 χ 10~ 6 second at room temperature. This long life time enables the compound to be used with an imaging gate to eliminate the background luminescence from the dye phosphorescence. In addition, because the complex has an intense and very broad absorption band at about 4500 A, it can be easily excited by the blue 4579 A line of the argon ion laser to produce emissions in the orange to near-infrared region of the spectrum at about 6100 A. This shift of about 1500 A is significantly greater than the typically observed shift between excitation and fluorescence. The ob served luminescence, therefore, is at much longer wavelengths than typi cally encountered from background flu orescence under blue-light excitation, and permits barrier filtering to sepa rate the incident energy from the fluo rescent image of the latent fingerprint.
ber of target sequences is increased a billion times to attain analytical quantities. The PCR product is then analyzed by dot-blotting to determine the genotype of the amplified DNA. An advantage of this method is that no electrophoretic separation is needed—all the DNA produced is identical. George F. Sensabaugh and Cecilia von Beroldingen of the Forensic Science Group at the University of California, Berkeley, working with Henry Erlich and Russell Higuchi of Cetus, have recently used the HLA DQ a system for PCR typing of the DNA from the roots of single human hairs. In the HLA DQ a system, there are six possible alleles that can combine to form 21 different genotypes, each with a particular popula tion frequency. At present, the HLA DQ a system is the only one that has been used in forensic analysis, November 20, 1989 C&EN
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PCR specifically amplifies a small region of DNA _
- Cycle 2-
Double-stranded DNA, up to 500 million base pairs _ Targeted sequence, about 300 base pairs
-Cycle 1 Heat denaturation yields single-stranded DNA templates
Site-specific primers anneal to complementary base pairs in the DNA templates .
. . . and initiate polymerasecatalyzed synthesis of double-stranded DNA along the template resulting in two copies of the targeted sequence, each identical to the original. pÉÉMHWpl | Ι 1^ΓΓ;Ι
Cycles 4-25 At least 105-fold increase in DNA In the first cycle of polymerase chain reaction (PCR) analysis, the DNA to be amplified is denatured with heat. Next, two oligonucleotide primers (labeled A and Β above)—sequences of nucleic acids 20 to 30 bases long that are complementary to the DNA flanking the target DNA region—are bound to the denatured flanking sequences, one on each strand. The primers initiate synthesis of DNA strands complementary to the single-stranded DNA templates. This process is catalyzed by DNA polymerase, the same enzyme that catalyzes the replication of DNA in the living cell. The target sequence is part of the newly synthesized DNA. This process is repeated 20 to 30 times. In each cycle, the copies of the original DNA are replicated as well as the original DNA. The copies of the copies include only the target sequence, whereas copies of the original DNA extend beyond the target sequence. The number of copies of copies grows exponentially, while the number of copies of original DNA increases arithmetically because the original complete DNA template is not replicated. As a result, the reaction mixture eventually contains primarily copies of the target sequence. The target DNA is copied 2 2 0 to 2 3 0 times, resulting in up to 10 9 copies in just a few hours. In theory, only one cell is needed for PCR amplification, which means that DNA in extremely small samples can be replicated with this technique.
but it is certain that others will be available in the very near future. If enough other systems are devel oped, the combined population statistics will produce an even more meaningful genetic result that can be presented in court. PCR has a detection limit which is several orders of magnitude lower than the RFLP method, which re quires a certain threshold amount of high-molecular26 November 20, 1989 C&EN
weight DNA for analysis. Choosing between RFLP and PCR, however, involves a tradeoff between sensi tivity and specificity. The RFLP approach offers greater potential for individualization, but a larger sample is needed. The PCR approach now in use requires much less sample, but is less informative. In the future, DNA profiling will undoubtedly take advantage of both methods.
DNA analysis of evidence has been successfully introduced in court cases in England and the U.S. Scientists working in this area have been careful and methodical in establishing the fundamental scientific underpinnings of DNA techniques to convince the courts that their work is valid. Twenty-five states have now accepted DNA typing as evidence. Appellate courts in Florida and Maryland have affirmed the use of DNA as evidence in criminal trials, but there has been no other appellate-level scrutiny of DNA to date. In part, this is because the DNA evidence has generally been so convincing at the investigative stages that the defendant has typically entered a plea of guilty, thus shortstopping any further legal review of the reliability of the method. Also, only a short time has elapsed since DNA evidence was introduced into the court system in late 1987, and appellate decisions can take several years. The greatest concern from all quarters—forensic chemists, the judiciary, and the defense bar—is a false identification in which one DNA type will be mistaken for another. This concern is certainly not inappropriate, but appears to be without substance. Environmental conditions can in fact degrade and alter DNA, as they can alter most other biochemical materials. However, according to experts at the FBI lab and at other labs that do DNA profiling, including Michael Baird, geneticist, and Robert Shaler, biochemist, at Lifecodes in Valhalla, N.Y., and Daniel Garner, a biochemist at Cellmark in Germantown, Md., adverse treatment of DNA simply renders it unreadable; it doesn't cause it to mimic another DNA sequence. This is not to say that DNA typing is infallible; there are, in fact, cases on record of errors having been made. In each of these cases, the mistake seems to have been prompted by human error. But a mistake is a mistake for whatever the reason, and justice is not to be dispensed from a test tube. Samples have been mismarked, the patterns on an autoradiogram have been incorrectly interpreted, h u m a n probes have apparently been contaminated with bacterial sequences, and, in one instance, two samples were split for purposes of balancing a centrifuge and then subsequently recombined incorrectly. DNA typing is technically demanding, and the quality assurance practices to ensure the validity of laboratory procedures have not always been in place. Every laboratory currently engaged in DNA typing has now established quality assurance protocols which, it is hoped, will keep the specter of human error as low as possible. An example of a lack of quality control is the case of New York vs. Castro, a homicide case in which a woman and her two-year-old daughter were stabbed to death. A neighborhood handyman, Jose Castro, was charged with the slayings. In a 15-week pretrial hearing to review the admissibility of DNA typing, it became apparent that errors had been made in both the typing technique and the subsequent interpretation conducted by Lifecodes. Eric Lander, a mathematician and geneticist at Harvard University and Whitehead Institute, Cambridge, Mass., was one of the principal defense
witnesses in the Castro hearing. He pointed out that although the prosecution sought to introduce evidence that adjacent lanes on a Southern blot autoradiogram contained identical band patterns, these lanes did in fact have different numbers of bands. Extra bands that could not be identified were dismissed by Lifecodes as being from nonhuman sources, but other samples that contained too few bands for a proper match were reported to contain additional bands. This is a question of the analyst not seeing something that was there, or seeing something that wasn't there. This is deplorable, but it is perhaps not the most serious issue. Of more concern, Lander pointed out, was the inability of classical population genetics to adequately describe the population in this particular case. Polymorphic alleles are not scattered at random through real human populations. Subgroups do in fact exist, such as among the Hispanic population of New York City, and profound deviations from classical population genetics may be encountered. The legitimate criticisms enunciated by Lander with respect to population genetics are not fatal to DNA typing, but do point out the need to interpret DNA typing with considerable diffidence and with a thorough understanding of the populations involved. That is, if the statistics invoked in a particular case are inappropriate to the population under consideration, serious errors in determining the probability or the significance of a "match" could result. It should be recognized, however, that this is by no means unique to DNA typing; this caveat applies also to the
The series of bands produced on x-ray film by the RFLP technique is incredibly rich in information. The bands in adjacent vertical lanes on the film can be compared to determine whether the DNA from two or more samples matches. If the bands match, the DNA is probably from the same person. Unless DNA from identical twins is being compared, the probability that two matching bands are not from the same person is as low as 1 in 1019 November 20, 1989 C&EN
27
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Special Report conventional analysis and interpretation of isoenzymes, serum proteins, and blood group antigens. In the Castro case, the trial judge threw out part of the DNA typing evidence—the inclusionary evidence, but not the exclusionary evidence—while at the same time expressing the court's confidence in DNA typing in general. Stated differently, the court said that DNA typing does in fact meet the test for reliability, but that in this isolated case some of the techniques were not performed correctly. "The testing laboratory failed in several major aspects to use the generally accepted techniques and experiments for obtaining reliable results, within a reasonable degree of scientific certainty," Justice Gerald Sheindlin wrote. (Incidentally, on Sept. 15, Castro pled guilty to murder and admitted that the blood on his watch was that of the victim— the same result that Lifecodes had reported after performing its flawed RFLP test on this blood.) Every new technique, of course, has its teething problems. DNA typing will be no different. Carelessness on the part of the analyst may from time to time occur, and, additionally, special problems may be encountered with contaminated samples that will cause practical difficulties. Unlike the clinical samples that are dealt with in DNA diagnostics, forensic samples may have been scraped off a sidewalk or from soil, or recovered from some equally dirty surface. It is virtually certain that such samples will be contaminated with bacterial DNA. In addition, some forensic samples may be exceedingly limited. For example, blood shed during the beating of a human being is frequently distributed by the force of the injury as a very fine aerosol in which no single bloodstain represents more than a small fraction of a microliter. When the blood of more than one person is comingled, the analyst may or may not be justified in pooling the stains for analysis. In short, the sampling and interpretation will necessarily require judgment calls on the part of the analyst that are unique to forensic, as opposed to clinical, practice. But given the gravity of a mistake within a forensic context, where a person may lose his or her liberty or even life, it is in the interests of all concerned to develop appropriate standards for both the analytical and interpretational aspects of DNA typing. Lander and others are advising the Congressional Office of Technology Assessment as it develops a report concerning DNA typing. Additionally, and with good reason, the defense bar is clamoring for the establishment of guidelines for DNA typing. The National Academy of Sciences and the National Institute of Justice of the U.S. Department of Justice have both been urged to convene expert panels to consider the matter. In a meeting, of forensic DNA workers at the FBI Research & Training Facility at Quantico, Va., this past June, a working group was selected to specifically address the need for objective standards, the necessity for internal controls, and quality assurance protocols. A preliminary draft of these recommendations has now been published in the FBI's Crime Laboratory Digest. Although the investigative interest in DNA typing is generally directed toward comparing two samples 30 November 20, 1989 C&EN
to determine if they could have come from the same person, the exclusionary use may be just as valuable, or perhaps even more so. According to John Hicks, director of the FBI laboratory, in the first 45 DNA cases submitted in connection with sexual assault offenses, DNA typing exonerated seven of those 45 individuals. The FBI laboratory is now accepting a considerable amount of DNA casework. It has already received several hundred specimens from its own agents and from police departments around the U.S. In addition, Lifecodes and Cellmark Diagnostics have been offering court testimony on DNA typing since the fall of 1987. Already, a number of other forensic laboratories across the nation are engaged in this work. The newly established U.S. Fish & Wildlife Service laboratory in Ashland, Ore., will use DNA technology in the enforcement of laws pertaining to endangered animal species. DNA typing will not significantly lower the crime rate in America. At most, it will simply add to the quality ethic that pertains to the process by which guilt is decided. But then, that is quite a lot in itself. The criminal justice system needs all the help that it can get. D
John I. Thornton is a professor of forensic science at the University of California, Berkeley. He received his bachelor's and master's degrees and his doctorate, all in forensic science, at Berkeley and he has taught there since 1970. He is a former president of the California Association of Criminalists, a member of the California Association of Cnme Laboratory Directors, and a former chairman of the Criminalistics Section of the American Academy of Forensic Sciences. He has taught also at the University of New. Mexico School of Medicine and in the People's Republic of China. Prior to his present teaching and research position, he worked for nine years in an operational crime laboratory in California. Thornton has written more than 150 publications in the area of forensic science. His current research interests are principally in the area of trace microanalysis, and most recently he has been engaged in the characterization of nitrated derivatives of diphenylamine in gunshot residues under a grant from the National Institute of Justice. Thornton wishes to thank Malcolm McGinnis for his contributions to the preparation of this article. Reprints of this C&EN special report will be available in black and white at $5.00 per copy. For 10 or more copies, $3.00 per copy. Send requests to: Distribution, Room 210, American Chemical Society, 1155—16th St., N.W., Washington, D.C. 20036. On orders of $20 or less, please send check or money order with request.
