POLYMERASE CHAIN REACTION BASIC THEORY AND APPLICATION IN FORENSICS PCR is one of the most widely used tools in Molecular biology. Perhaps 100’s of ‘recipe’s where variants of this technique have been employed Commonly referred to frequently on television Biologists should know how to explain a technique that is so commonly referred to in the media to the lay public. Everyone who has ever turned on a television now knows that forensic scientists can get a positive identification from just a speck of blood. While we look different, we vary on average only one base in 1200. How then can we accurately predict the identity of a suspect. Why is this technique so much more reliable than eye witness accounts?

POLYMERASE CHAIN REACTIOBASIC THEORY AND APPLICATION IN FORENSICS This figure comes from wikipedia which has a good review of the subject of PCR

Designing primers to amplify a DNA fragment Left primer: 5’cgagaattcATGGCCCCC3’ RIGHT Primer 5’ TCGAAGCTTTTACAAAGAGTC3’ cgagaattcATGGCCCCCA ATCCTGCTGCTGCTCCTGTGGAGCTGCGTCGTCTCAACTTCGCGTTCGCCAAGGTGACAATCGGTGGACA CCTTGTGAACCGATTGCAACACGACCCAGTTAACGGGGACCCCCAACAAGAGGACGATCGCGTCCTACGT GACGTGGACCTCACGCTGCAGTCTGGCCAACGAGTGCTGGTGGTGGGTGGCAATGGGGCTGGCAAGAGCA CGCTGTTGAGCATCCTGGCTGGGAAACACTTGACTGCGGACGACACGGCGCTGGTCTTTGGTCGGGACAG TTTCCGAGATACGACGCTCAATGCGTTGAGGACCTTCGTGAGCGCAGATTGGGGCCAACGCGCCGTGGCC TTCGCGTCGCATGCCATGGCGTATTCGGCCGATATGGCGGTGGAGGAGATGATGGTGAAGCTGCAGAGTG AACACCCGGAGCGACGCGTGAAGCTGCTGAAGGTGCTGCGGATCGACCCCAAGTGGCGTCTGCATCGCCT GTCGGACGGCCAGCGGCGTCGTGTGCAGCTGTTCCTGGCGTTGCTGCGACCGTCGCAGTTGATCATCCTG GACGAGGTGCTGGGCATGCTCGACATCATCTCGCGAGAGAACGTGTTGGCCTTCTTGAAGGAGGAGACGG AGACCAGGCAGGCCACGGTACTGCTGGCCACGCACATTTTCGACGGCACGGACGTCTGGGCGTCTCATGT GCTGTACATTCGTCGCGGCGGAGTGGGTTTCTATGGCCCCATTGAGCAGTGCACTGACGGCCAGAAGGTC CCGATGTACAAGGCGGTGGAGAACTGGCTGCGTACTGAATTGGCTGAGGACGACCGCGTGGAGACTGAGG CCGTTGGTGCGAGCGGGGAGTTCGATCTGGCCAACGCTCAGAACCGGGCTGGCGGATACGCTGCTGGTCG ACTGGGTGGCGTCGATATCGACTCTTTGTAAaagcttcga Primers need to be long enough so that they can bind specfically to a single locus and no other location. The left primer is usually 18-23 bp and is an exact copy of the DNA sequence. . The right primer must bind to the top sequence so it is synthesized as a reverse complement to the sequence shown in red. End Sequence 5’GACTCTTTGTAAaagcttcga3’ Reverse complement 5’ TCGAAGCTTTTACAAAGAGTC3’

Short Tandem Repeats (STRs) AATG 7 repeats 8 repeats The same set of primers will amplify both the 7 and 8 tetranucleotide repeats. How many combinations of products would you expect? the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length (one PCR product) Heterozygote = alleles differ and can be resolved from one another, (2 PCR products.)

SHORT TANDEM REPEAT SEQUENCES http://www.cstl.nist.gov/div831/strbase//intro.htm Tandemly repeated DNA sequences are widespread throughout the human genome and show sufficient variability among individuals in a population that they have become important IN human identity testing. Minisatellites (variable number of tandem repeats, VNTRs) have core repeats with 9-80 bp, while microsatellites (short tandem repeats, STRs) contain 2-5 bp repeats. The forensic DNA community has moved primarily towards tetranucleotide repeats, which may be amplified using the polymerase chain reaction (PCR) with greater fidelity than dinucleotide repeats. The variety of alleles present in a population is such that a high degree of discrimination among individuals in the population may be obtained when multiple STR loci are examined.

http://www.cstl.nist.gov/strbase/ This is a web site showing the various loci and primer sets that can be used to identify idividuals.

195 bp 170 bp TCAT repeat unit Different primer sets produce different PCR product sizes for the same STR allele

Sex determination with Amelogenin protein Forensic PCR depends on primers that will reliably amplify a specific region of the genome that is also known to be highly variable across individuals. B Sex determination with Amelogenin protein http://en.wikipedia.org/wiki/Amelogenin The amelogenin protein is present in both the X and y chromosome but the size is different between the x chromosome and the y chromosome. When primers are used to amplify this intron, females will have two bands and males will have only one band. Protein found in tooth enamel Intron 1 is 106 bp on X chromosome and 112 bp on Y chromosomes Primers specific for the intron can determine sex.

13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 TH01 D8S1179 D5S818 VWA FGA D7S820 CSF1PO The loci that can be amplified with specific primers are scattered across the genome. AMEL D13S317 AMEL D16S539 D18S51 D21S11

Multiplex PCR Over 10 Markers Can Be Copied at Once Sensitivities to levels less than 1 ng of DNA Ability to Handle Mixtures and Degraded Samples Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges One tube 10 primer pairs, one set of template DNA. Each of the primers ar designed to only amplify a specific locus. Each primer pair is tagged with a particular color of fluorescent dye so that the products can be distinguished

Several companies provide PCR kits for forensic identification http://www.cstl.nist.gov/biotech/strbase/multiplx.htm

PCR fragments Are Separated by Capillary Electrophoresis Different gene primers have one of four fluorescent tags so bands can be associated with a specific locus.

ABI Prism 310 Genetic Analyzer capillary Syringe with polymer solution Autosampler tray Outlet buffer Injection electrode Inlet buffer

An Example Forensic STR Multiplex Kit AmpFlSTR® Profiler Plus™ Kit available from PE Biosystems (Foster City, CA) 9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction 100 bp 400 bp 300 bp 200 bp Size Separation Color Separation D3 FGA vWA 5-FAM (blue) D13 D5 D7 NED (yellow) A D8 D21 D18 JOE (green) GS500-internal lane standard ROX (red) Each individual will have one or two bands at each loci such as D3 depending on whether they are homozygotic or hetrozygotic for that locus. Alleles from the D3 loci can range in size from ~110bp to ~150 bp Alleles from the D5 loci can range in size from 120 bp to 160 bp What is the maximum & minimum number of bands that you would expect to see in a successful reaction.

Human Identity Testing with Multiplex STRs Simultaneous Analysis of 10 STRs and Gender ID AmpFlSTR® SGM Plus™ kit Two different individuals DNA Size (base pairs) amelogenin D19 D3 D8 TH01 VWA D21 FGA D16 D18 D2 Results obtained in less than 5 hours with a spot of blood the size of a pinhead probability of a random match: ~1 in 3 trillion

STR Allele Frequencies 5 10 15 20 25 30 35 40 45 6 7 8 9 9.3 TH01 Marker Number of repeats Frequency Caucasians (N=427) Blacks (N=414) Hispanics (N=414) A DNA profile may HINT at the potential ethnicity of an individual. A recent example is an “indian woman “ who learned from DNA analysis that her ancestors included Spanish emmigrants of Jewish decent. *Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

FORENSICS AND HOME PATERNITY TESTS http://www.dnatesting.com/resource/sampleTests.php Paternity testing is regrettably a business that has grown out of this technology. Children must have inherited the loci from mothers or fathers. The table shows results from 15 genetic loci. Due to a mutation during replication the probability of any locus in the child being different than the aprent is 1/1000, but the probability of two loci being different is 1 in a million. Therefore if the childs DNA fails to match the suspected factor at two loci, then the “father’s brother” is a more likely candidate of being the real biological parent!

Review questions In multiplex PCR, several primers are added to the same tube at once. How is it possible that you amplify only the products that you want? Aren’t you limited to just two primers? Why might this approach be better than separate PCR reactions?