Chapter 20.1-20.2.1: DQA1/PMChapter 18: Autosomal STR Profiling

Based on SNPs (single nucleotide polymorphisms) Variations in the human genome Single-base pair change originating from

spontaneous mutations Majority of human DNA polymorphisms 1.4 million identified Most are bi-allelic (e.g. AGT and ATT are

common alleles but ACT and AAT are not)

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Advantages: SNPs are abundant and can be used as

markers Can easily be amplified by PCR

▪ 50-100 bp in length No “binning” or statistical problems Useful for phenotyping

Disadvantages: Not very polymorphic (most are only

dimorphic)▪ Much lower level of discrimination than DNA

Fingerprinting or STR analysis3

Very important during late 1980’s and 1990’s in forensic labs (before discovery of STRs)

PCR-based: Allowed for analysis of trace evidence and

degraded samples Loci:

HLA-DQA1 (Average Pm = 1/20) Polymarker (Average Pm = 1/164) Combined average Pm = 1/3,300

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Methods to detect SNPs: DNA sequencing (labor-intensive) Allele-specific oligonucleotide (ASO)

hybridization assays (fast and easy)▪ ASO probes hybridize to their complementary DNA

sequences to distinguish known polymorphic alleles▪ Steps:

▪ DNA extraction▪ PCR amplification across the SNP site using biotinylated

primers▪ Denaturation of DNA hybridization to immobilized probes▪ Colorimetric detection

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Like VNTRs, are tandem repeats Length of repeat motif is less than 10 bp Also known as “microsatellites” Block is usually less than 500 bp Advantages:

Lots of them (more than 100,000 in human genome) Very polymorphic Block is small enough for PCR amplification

▪ Good for trace evidence and degraded DNA▪ Amplicons can be separated on high resolution

polyacrylamide gels

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Repeat Unit Length Dimeric (CT), Trimeric (CAT), Tetrameric

(CTTG) ... High degree of polymorphism

▪ Mutation “hot spots”▪ Located in intergenic DNA

Micorvariants (e.g. 15.2) Population Match Probability

Best STR systems:▪ Highly polymorphic STRs▪ Relatively equal frequency distribution at all loci▪ Unlinked 14

STR multiplex system in U.K.- 4 loci1995- first national database

established in U.K. 6 STR loci + amelogenin (4 loci added later)

1997- first database in U.S. CODIS FBI 13 core loci + amelogenin 2 more now added for a total of 15

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Loci are amplified using fluorescent dye-labeled primers

Separated using polyacrylamide electrophoresis

Detection: Wavelength of fluorescence Time to window Amplitude of signal

Results in an electropherogram Size of each amplicon determined by

comparison to internal size standard (ROX, LIZ)

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Relative fluorescent units (rfu’s)

Time since injection = amplicon length

Mutations at STR core repeat regions During embryogenesis During spermatogenesis

DuplicationsPrimer binding site mutationsAmplification artifacts

Allelic drop out , allelic drop in, stutterElectrophoretic artifacts

Pull-up, dye blobs, and spikes

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Degraded DNA MiniSTR multiplex kits

Low-copy Number DNA (LCN) < 100 pg of DNA

Mixtures Sexual assault cases Mixture interpretation

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SWGDAM & DNA Commission of the ISFG: Inclusion (Match)

▪ Calculate Pm ▪ Sometimes challenged in Court (especially mixtures)

Exclusion▪ No calculation needed▪ Sometimes challenged in Court (especially mixtures)

Inconclusive▪ Multiple interpretations may be possible▪ Often challenged in Court

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