Introduction to Forensic Sciences 2nd Edition phần 8 pdf

which are degraded or in some way made smaller by actions of physical or environmental agents or age. Because theoretically it is possible to multiply single strands of DNA to essentially millions of copies of that single sequence, PCR is extremely sensitive, and from 1 to 5 ng of DNA can be successfully typed using the process. 23 Figure 11.10 and Table 11.6 compare the differences between conventional RFLP/DNA analysis and PCR/DNA analysis. Figure 11.9 The polymerase chain reaction: Multiple copies of specific DNA segments or genes were originally produced by cloning, cutting out the segment wanted and inserting it into a host cell which would, as it reproduced, make copies of the inserted gene along with its own DNA. Now DNA may be copied enzymatically using a temperature-insensi- tive DNA copying enzyme or polymerase. Starting with double-stranded DNA, the specific site to be amplified is targeted by using primers which flank the target site and act as anchors for the synthesis of a new DNA strand. (A) The first step of a cycle involves melting the DNA to expose the nucleotides of each strand allowing the primers to bind. (B) Next the temperature is lowered and the polymerase enzyme facilitates the synthesis of two new DNA strands using the old strands as templates. (C) These two double-stranded DNA molecules are melted or denatured in cycle 2 to begin the process anew. It is called a chain reaction, because at each cycle the number of previously existing DNA molecules is doubled. C A, B A ©1997 CRC Press LLC Restriction Fragment Length Polymorphism (RFLPs) of Variable Number of Tandem Repeats (VNTRs) Are the Basis for the Original DNA Profiling At many locations throughout the human genome, short and long sequences (two to over several hundred base pairs) of DNA bases are repeated over and Figure 11.10 Comparison of RFLP (Southern-blot) and PCR (amplified) DNA identifica- tion tests: The power of forensic tests depends on their ability to include or exclude individuals as contributors of evidentiary samples, but the application of particular tech- niques depends on the number of tests required to produce odds of exclusion which suggest that the match means that the evidentiary sample came from the identified individual. Selection of a test or set of tests also depends on the condition of evidence. Fewer RFLP tests than PCR tests are needed to produce a given probability, but larger sample sizes are required for RFLP tests. In the future, PCR-based sequence-like or sequence-based tests may eliminate this difference. Table 11.6 Comparison of Two Analysis Methods for Forensically Applicable DNA Loci PCR VNTR/RFLP Aged, Degraded, or intact DNA Intact large sequences 100–3000 bp 500 to 23,000 bp 1–25 ng Greater than 50 ng Fewer alleles Large number of alleles 2- to 3-day analysis time 6- to 8-week isotopic analysis More loci needed for high discrimination 4 to 5 loci needed for high discrimination High discrimination High discrimination 8–22 alleles 250–1,000 alleles ©1997 CRC Press LLC over again. 15,24 They are said to repeat in tandem, meaning they repeat con- tinually in a chain-like sequence. The number of these repeating units is highly variable, so that most people have differing amounts of these repeat units inherited from their mother and father (see Figure 11.11). When the length of the repeat units inherited from each parent will be different, this condition is known as heterozygosity. The existence of these repetitive sequences is very important, as they are being used to locate certain genes in our chromosomal structure, and also some have been linked to genetic diseases such as fragile X syndrome and myotonic dystrophy. 25 They are also excellent sites to use in assessing differences among humans. 26 By use of a technique such as RFLP the forensic scientist is able to quantitate the lengths of these VNTRs and use them to identify with high precision and accuracy the identity of an individual. Because there are a limited number of these variations at any one site, different individuals can share similar size VNTRs. It then is not possible to determine the source of a sample from use of one VNTR locus (spot on a chromosome). If, however, many different VNTRs are used, a profile of an individual is created with a very high discrimination power (see Figure 11.12). 27, 28 There are several ways to determine the length of the VNTR. One is by using a restriction enzyme previously described to “cut” the VNTR out of the piece of DNA and then to separate the pieces using elec- trophoresis. Another way is the use of the PCR process to copy or amplify the VNTR pieces of less than 3000 bp and then separate them by electro- phoresis. 29 This does not involve the use of restriction enzymes but the use of PCR primers. Figure 11.11 Example of a VNTR (variable number of tandem repeat): Genomic DNA, all the DNA in each cell, contains many sections which are repetitive. These often vary from individual to individual, because the number of repeats is different. Because of this, the length of the repeat DNA will vary when this section of DNA is cut out and visualized or amplified and visualized. The “repeat units” can be as small as a single base pair to many hundreds of base pairs making up a repetitive sequence. The sequence may be many thousands or millions of “repeat units”. This kind of DNA has been referred to as “junk” DNA in the past, but new evidence may show that it may not be junk after all. Thus, the sequence made up of “repeat units” from our father may differ from the sequence from our mother. When there is a difference between genetically defined multiple forms of a particular character, it is called polymorphism. ©1997 CRC Press LLC Other Types of VNTRs and Sequence Polymorphic Areas of Nuclear and Mitochondrial DNA are Also Used to Profile DNA in Forensic Cases Small, amplified DNA segments are currently used in sequence-type analysis (mtDNA and MVR), fragment size analysis (STR, AMPFLP), or dot-blot analysis (DQalpha and amplitype PM™). 7,8 In sequence-type analysis, the amplified DNA fragments may be sequenced directly after amplification using any of the sequencing methods, or the amplified fragments may be separated to produce a ladder which resembles a sequencing ladder. Direct Figure 11.12 Four DNA probe composites of autorads containing samples from suspect, victim, and evidence with a final frequency of occurrence of 1 in 3,400,000,000: The four autoradiograms or autorads in this figure depict matches between the suspect’s DNA patterns and those produced by the DNA extracted from the evidence. Since these four pattern matches are first made visually and subsequently by computer sizing, the examiner has estimated the odds of someone other than the suspect also producing a matching DNA pattern for each of the four probes. These are the numbers under each of the four autorad diagrams: 1 in 200, 1 in 303, 1 in 125, and 1 in 450. For the first match, using probe #1, the odds of finding another unrelated person with the same DNA pattern as the evidence are 1 in 200; this means that 1 / 2 of 1% of all humans will produce this pattern. There are, however, approximately 5 1 / 2 billion people on this planet; so, we must consider the fact that these odds of 1 in 200 mean that approximately 27 million unrelated people will also produce DNA patterns which match the evidence. DNA pattern matching power depends on the use of three to five different probes to identify independent DNA segments. The independence has been statistically verified and the examiner is, therefore, able to combine the odds for each probe to estimate the odds of finding an unrelated individual, other than the suspect, who would also produce matches on all four autorads. This is done by multi- plying 200 × 303 × 125 × 450 to produce composite odds of approximately 1 in 3.4 billion. These odds suggest that slightly more than 1.6 people on the planet who are unrelated to the person who produced the evidentiary pattern will match on all four of the autorads. ©1997 CRC Press LLC sequencing is most frequently done with the highly polymorphic control region or D-loop of mitochondrial DNA, and approximately 400 bp are sequenced. 9 This is a valuable technique for identification, because all mater- nal relatives are expected to have identical mtDNA; however, it is, for the same reason, less discriminating because maternal relatives are indistinguishable. 30 Minisatellite Variant Repeats (MVR) MVR (minisatellite variable repeat sequencing) uses the fact that some VNTR repeats have internal polymorphisms which may be used as terminators in much the same way that Sanger sequencing uses dideoxynucleotides to ter- minate polynucleotides in synthesis. These are then separated on a gel, and the sequence is read directly off the gel. The minisatellites MS31 and MS32 are currently the only VNTRs being used for identification using MVR methods. 31 AMPFLP (Amplified Fragment Length Polymorphism) Represents Another Type of VNTR that Is Smaller than the VNTRs Used for RFLP In AmpFLP analysis (amflip, or amplified fragment length polymorphism), sample DNA is amplified using primers which flank a core repeat of approx- imately 10 to 20 bp (see Figure 11.13). 7 The fragment length polymorphism, like VNTR polymorphism, is based on the number of core repeats found in a particular allele. Here alleles are defined as DNA segments on homologous chromosomes with different numbers of repeats. For example, the AmpFLP inherited from the mother may have 74 repeats, while that inherited from the father may have 38 repeats. AmpFLP alleles are, however, smaller than VNTR alleles. They range in size from 100 to 1000 bp, while VNTR alleles range from 200 to more than 20,000 bp. Most VNTR alleles are too long at this time to be amplified by PCR, and the ends or termini of VNTR alleles are defined by restriction enzyme cut sites which flank the tandemly repeated core sequences. Long PCR may change this limitation. Long PCR may be able to amplify more than 20,000 base pairs of DNA in the very near future. AmpFLP loci are selected for analysis because the size range of core repeats is efficiently amplified by PCR; consequently, the termini of these AmpFLP alleles are defined by primer sites, not by restriction sites. These differences of size explain, in large part, the fact that VNTR loci have an enormous number of alleles continuously distributed over the size range, while AmpFLP loci have an approximately discrete number of alleles, usually in the range of 5 to 25. ©1997 CRC Press LLC Following amplification, the DNA sample is separated on a gel, usually polyacrylamide, and the amplified fragments are visualized with silver stain or fluorescent dyes. As in VNTR analysis, size ladders are included on the analytical gels. In AmpFLP analysis, however, alleles are treated as discrete units which allows visual comparison of alleles with the ladder alleles, unlike VNTR analysis which requires computer sizing. Figure 11.13 Examples of various repeat polymorphisms in the human genome: Genomic DNA, all the DNA in each cell, contains many sections which are repetitive. These often vary from individual to individual, because the number of repeats is different. Since the number of repeats is different, the length of the repeat DNA will vary when this section of DNA is cut out and visualized or amplified and visualized. (A) VNTRs have repeat lengths or cores which range from 9 to 40 bp, depending on which specific gene is being examined. These VNTRs are isolated and visualized for pattern comparisons by restriction enzyme digestion and DNA probing. VNTRs are the genes first used by forensic labs to produce “DNA fingerprints” by combining patterns over four to six genes. Although VNTRs are highly polymorphic, this testing methodology is expensive and time consuming. Two additional repeat length polymorphism tests have been introduced to reduce costs and testing time. Rather than cutting and probing, these tests amplify specific repeats, and the amplified sequences are visualized without probing. (B) AMPFLP (amplified fragment length polymorphisms) have cores which range from 8 to 16 bp. (C) STR (short tandem repeat) cores range from 4 to 6 bp. ©1997 CRC Press LLC Short Tandem Repeats (STR) Represent a Very Small VNTR Another type of amplified repeat analysis is STR, or short tandem repeat analysis. It is very similar to AmpFLP analysis, but the repeat sequences are shorter still (4 to 6 bp). Additionally, a number of STR loci may be amplified and separated simultaneously, a technique known as multiplexing. This increases the discriminatory power of STR analysis, while decreasing the work and time involved in the analysis. There are approximately 4.0 × 10 8 STR loci dispersed throughout the human genome. STRs consist of small numbers of repeat units, usually three, four, or five repeats, which are from 50 to several hundred base pairs in length. Much effort is being made at present to shorten the time of analysis of STRs and AmpFLPs, as well as to increase the number of STR and AmpFLP types analyzed. Several types can be analyzed at the same time and on the same gel with sophisticated hardware and novel tags attached to the DNA. One of these attempts has been the use of fluorescent-tagged STRs and AmpFLPs. Dr. Ron Fourney and Dr. C.J. Fregeau of the Royal Canadian Mounted Police have been instrumental in developing this technology (see Figure 11.14). The First Two PCR Systems to Find Wide Acceptance in the Forensic Community: HLA DQ-Alpha and Polymarker PM Two discrete allele systems which are not based on repetitive core sequences are currently used with PCR for identification. The first uses a portion of the human major histocompatibility complex (MHC) as a PCR substrate. The MHC is the complex gene or supergene which plays a role in tissue and organ differentiation and is one of the genes which must be matched in organ transplanting. This is the DQalpha locus, which is employed in a dot-blot format. Probes for the DQalpha alleles are fixed to membranes as dots. The sample DNA is amplified, and allelic identification is achieved by allowing the amplified sample to bind to the appropriate dots on the membrane. The bound sample is then visualized using a conjugated enzyme and dye. Then, the allele is identified as having bound to its complement on the membrane (see Figure 11.15). The second discrete allele system is amplitype PM™. It consists of certain probes bound to a strip as in the case of HLA DQalpha. The loci or types are as follows: HLA DQ-alpha (separate strip), LDL receptor (LDLR), glyco- phorin A (GyPA), hemoglobin G-gamma globin (HBGG), D7S8, and group specific component (GC). The advantage of this typing system is that one ©1997 CRC Press LLC Figure 11.14 The evolution of DNA typing in North America: This montage represents the evolution of DNA typing in North America from the (a) initial single locus DNA typing profile (D1S7) followed by PCR based methods including (b) AMP-Flaps (D1S80), (c) mini- satellite variant repeats ([MVRs] D1S8) and (d) fluorescent-tagged short tandem repeats (STRs, bottom: HumCD4, yellow; HumFABP, blue; HumACTBP2, green; ABI Genescan™ 2500 marker, red). Contributed by the Biology Research and Development Support Unit and Richard Musgrave of the Forensic Photo Unit, Royal Canadian Mounted Police. (Figure courtesy of Eaton Publishing Company.) See color plate. ©1997 CRC Press LLC [...]... al., J Forensic Sci., 31, 409 (1 986 ) 21 E Southern, J Mol Biol., 98, 503 (1975) 22 R K Saiki et al., Science, 260, 1350 (1 985 ) 23 H Erlich, Principles and Applications for Forensic Amplification (Stockton Press, Stockton, 1 989 ) 24 J C S Fowler et al., J Forensic Sci., 33, 1111 (1 988 ) 25 H G Brunner et al., Am J Hum Genet., 52, 1032 (1993) 26 A J Jeffreys, V Wilson, S L Thein, Nature, 314, 67 (1 985 ) 27... M Stoneking et al., Nature Genetics, 9, 9 (1995) 11 R E Gaensslen, Sourcebook in Forensic Serology, Immunology, and Biochemistry (U.S Department of Justice, National Institute of Justice, Washington, D.C., 1 983 ) 12 R Saferstein, Ed., Forensic Science Handbook, vol II (Prentice Hall, Englewood Cliffs, NJ, 1 988 ) 13 C A Villee et al., Biology (Saunders College Publishing, Philadelphia, 2nd ed., 1 989 )... Saferstein, Ed., Forensic Science Handbook, vol I (Prentice Hall, Englewood Cliffs, NJ, 1 982 ) 6 L Presley, personal communication, FBI Laboratory (1994) 7 R Saferstein, Ed., Forensic Science Handbook, vol III (Regents/Prentice Hall, Englewood Cliffs, NJ, 1993) 8 K B Mullis, F Ferre, R A Gibbs, Eds., The Polymerase Chain Reaction (Birkhauser, Boston, 1994) 9 M M Holland et al., J Forensic Sci., 38, 542 (1993)... An Introduction (Stockton Press, New York, 1990) 28 B Budowle et al., J Forensic Sci., 35, 530 (1990) 29 D Botstein et al., Am J Hum Genet 32, 314 (1 980 ) 30 J C Avise, in Ann Rev Genet., 25, 45 (1991) 31 A J Jeffreys and R Neumann, V Wilson, Cell, 60, 473 (1990) 32 J Zonderman, Beyond the Crime Lab (John Wiley & Sons, New York, 1990) 33 A J Jeffreys and J F Y Brookfield, R Semeonoff, Nature, 317, 81 8... late 180 0s.32 All of the characteristics he used ©1997 CRC Press LLC Table 11 .8 The HLA DQalpha Genotype Frequencies in Three Populations DQa, Genotype Caucasian (N = 737)a Black (N = 589 ) Hispanic (Composite) 0.019b 0.045 0.024 0.031 0.039 0. 085 0.053 0.026 0.049 0.072 0.113 0.0 08 0.014 0.016 0.054 0.020 0.047 0.064 0.042 0.106 0.072 0.020 0.090 0.007 0.032 0.022 0.0 78 0.065 0.017 0.053 0.071 0. 180 0.003... each character We may employ basic principles of the science of genetics to estimate frequencies Class vs Individual Characteristics: The Cornerstones of Identification in Forensics that Brings the Value of Serological Evidence into Perspective when Understood by Laymen There are three categories of evidence submitted to a crime laboratory: class, individual, and an intermediate category where class evidence... 1 987 ) 15 R J MacIntyre, Ed., Molecular Evolutionary Genetics (Plenum Press, New York, 1 985 ) ©1997 CRC Press LLC 16 M Singer and P Berg, Genes and Genomes (University Science Books, Mill Valley, CA, 1991) 17 B Budowle et al., Crime Lab Dig., 18, 9 (1991) 18 Cellmark Diagnostics, DNA Fingerprinting, The Ultimate Identification Test, Germantown, MD (1992) 19 E Kanter et al., J Forensic Sci., 31, 403 (1 986 )... material to run 20 to 40 tests These limitations confined serologists to match conclusions phrased in “failure to exclude” terms until the mid 1 980 s In 1 985 , Alec Jeffreys introduced the use of DNA for the identification and exclusion of humans; he dubbed the multilocus technique he used “DNA fingerprinting”.26,33,34,35 As we have emphasized, both classic fingerprinting introduced by Francis Galton in the... Harrington, Eds., Forensic DNA Technology (Lewis Publishers, New York, 1991) 2 K E Boorman and B E Dodd, An Introduction to Blood Group Serology (Churchill Livingstone, Edinburgh, 4th ed 1970) 3 O Prokop and G Uhlenbruck, Human Blood and Serum Groups (Wiley Interscience, New York, 1969) 4 B J Culliford, The Examination and Typing of Bloodstains in the Crime Laboratory (U.S Department of Justice, Washington,... multiplying allele or gamete frequencies to predict genotype or offspring frequencies This will be a valid procedure only if we can show to a reasonable approximation that some alleles are not found together in offspring more or less frequently than they would be if drawn at random The situation is analogous to coin tossing or die rolling Two fair, two-sided coins may be tossed and the percentage of times . alleles 2- to 3-day analysis time 6- to 8- week isotopic analysis More loci needed for high discrimination 4 to 5 loci needed for high discrimination High discrimination High discrimination 8 22 alleles. translation to amino acids to form proteins. Ultimately, then, it is reasonable to expect that DNA sequence information will be used by forensic scientists. Currently the DNA from the mitochondria. 0.039 0.022 0.0 48 1.1,4 0. 085 0.0 78 0.079 1.2,1.2 0.053 0.065 0.023 1.2,1.3 0.026 0.017 0.034 1.2,2 0.049 0.053 0.034 1.2,3 0.072 0.071 0.073 1.2,4 0.113 0. 180 0.107 1.3,1.3 0.0 08 0.003 0.004 1.3,2