Chapter 20 1 20 2 1 dqa1 pm chapter 18 autosomal str profiling
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Chapter 20.1-20.2.1: DQA1/PM Chapter 18: Autosomal STR Profiling. Forensic Biology by Richard Li. DQA1-PM PCR -based Typing. Based on SNPs ( s ingle n ucleotide p olymorphisms) Variations in the human genome Single-base pair change originating from spontaneous mutations

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Forensic Biology by Richard Li

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Chapter 20 1 20 2 1 dqa1 pm chapter 18 autosomal str profiling

Chapter 20.1-20.2.1: DQA1/PM

Chapter 18: Autosomal STR Profiling

Forensic Biologyby Richard Li


Dqa1 pm pcr based typing

DQA1-PM PCR-based Typing

  • 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)


Basic characteristics of snps

Basic Characteristics of SNPs

  • 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


Forensic applications

Forensic Applications

  • 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


Forensic applications1

Forensic Applications

  • 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


Str typing basics

STR Typing: Basics

  • 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


Characteristics of str loci

Characteristics of STR Loci

  • 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


Str commonly used

STR Commonly Used

  • 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


Forensic str analysis

Forensic STR Analysis

  • 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)


Forensic biology by richard li

Relative fluorescent units (rfu’s)

Time since injection = amplicon length


Factors affecting genotyping results

Factors Affecting Genotyping Results

  • 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


Genotyping of challenging forensic samples

Genotyping of Challenging Forensic Samples

  • Degraded DNA

    • MiniSTR multiplex kits

  • Low-copy Number DNA (LCN)

    • < 100 pg of DNA

  • Mixtures

    • Sexual assault cases

    • Mixture interpretation


Interpretation of results

Interpretation of Results

  • 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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