1. Chapter 20.1-20.2.1: DQA1/PM Chapter 18: Autosomal STR Profiling

2.  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) 2

3.  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 analysis 3

4.  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 4

5.  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 5

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11.  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 11

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14.  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

15.  STR multiplex system in U.K.- 4 loci  1995- 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 15

16.  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) 16

17. 17 Relative fluorescent units (rfu’s) Time since injection = amplicon length

18.  Mutations at STR core repeat regions  During embryogenesis  During spermatogenesis  Duplications  Primer binding site mutations  Amplification artifacts  Allelic drop out, allelic drop in, stutter  Electrophoretic artifacts  Pull-up, dye blobs, and spikes 18

19.  Degraded DNA  MiniSTR multiplex kits  Low-copy Number DNA (LCN)  < 100 pg of DNA  Mixtures  Sexual assault cases  Mixture interpretation 19

20.  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 20