1. 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 How then can we accurately predict the identity of a suspect. Why is this technique so much more reliable than eye witness accounts?

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

3. 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 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’

4. 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.)

5. SHORT TANDEM REPEAT SEQUENCES
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.

6.
This is a web site showing the various loci and primer sets that can be used to identify idividuals.

7. 195 bp
170 bp
TCAT repeat unit
Different primer sets produce different PCR product sizes for the same STR allele

8. 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
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.

9. 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

10. 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

11. Several companies provide PCR kits for forensic identification

12. 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.

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

14. 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.

15. 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

16. STR Allele Frequencies5 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

17. FORENSICS AND HOME PATERNITY TESTS
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!

18. 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?