chapter 20.1-20.2.1: dqa1/pm chapter 18: autosomal str profiling
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Chapter 20.1-20.2.1: DQA1/PMChapter 18: Autosomal STR Profiling
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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
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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
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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
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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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