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Forensic Biology by Richard Li, with additions and edits by Ruth Ballard

Lecture 12: Autosomal STR DNA Profiling. Forensic Biology by Richard Li, with additions and edits by Ruth Ballard. Outline. STRs as DNA Markers Amplification by end-point PCR AmpF l STR Identifiler system Separation of a mplicons by CGE Size standard Allelic ladder

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Forensic Biology by Richard Li, with additions and edits by Ruth Ballard

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  1. Lecture 12: Autosomal STR DNA Profiling Forensic Biologyby Richard Li, with additions and edits by Ruth Ballard

  2. Outline • STRs as DNA Markers • Amplification by end-point PCR • AmpFlSTRIdentifiler system • Separation of amplicons by CGE • Size standard • Allelic ladder • Interpretation of STR profiles • Artifacts of CGE • Calculating random match probabilities • Low template DNA • DNA mixtures

  3. STRs as DNA Markers • STRs = short tandem repeats • Length of repeat motif is less than 10 bp • Also known as “microsatellites” • Block of repeated units (taken together) <500 bp • Forensic DNA profiling systems use • Tetranucleotide (e.g. TACA) • Pentanucleotide (e.g. GGCAT)

  4. STRs as DNA Markers • Allele defined as the number of repeats at the STR locus • E.g. GACA repeated 15 times in a row • # genotypes = (x2 + x)/2 • E.g. ABO system # alleles = x = 3; # genotypes = 6 • E.g. vWA; # alleles = x = 14; # genotypes = 105!

  5. Amplification by end-point PCR • Block is small enough for PCR amplification using primers flanking the repeated block • Good for trace evidence and degraded DNA

  6. Amplification by end-point PCR • Several commercial kits available for forensic STR profiling • Life Technologies: AmpFlSTR Identifiler Plus • 15 STRs + amenogelin (sex-typing locus) • We will use this kit in lab • Promega: PowerPlex 16 • 15 STRs + amenogelin (sex-typing locus) • Kits with even more loci now available (e.g. PowerPlex 21)

  7. Amplification by end-point PCR • AmpFlSTR Identifiler Plus kit contains: • Primer mix • 2 primers per locus = 32 for Identifiler • Master mix • dNTPs • Buffers and salts • Taq DNA polymerase • Allelic ladder (more on this later) • Only one reaction is needed to amplify all 16 loci • “Multiplex” system is faster than 16 separate reactions

  8. Amplification by end-point PCR • Primers are tagged with fluorescent dyes • The primers get incorporated into the amplicons, thereby labeling them

  9. Amplification by end-point PCR • Four different dyes used to label the amplicons from the 16 different loci (TABLE?) • FAM = blue • VIC = green • NED = yellow • PET = red • One dye used for size standard • LIZ= orange • More on this later

  10. 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)

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

  12. Factors Affecting Genotyping Results • Primer binding site mutations • Amplification artifacts • Allelic drop out , allelic drop in, stutter • Electrophoretic artifacts • Pull-up, dye blobs, and spikes

  13. 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

  14. Interpretation of Results • SWGDAM & DNA Commission of the ISFG: • Inclusion (Match) • Calculate RMP • 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

  15. Typical Report Wording Charles Anderson Gibs is included as a contributor to the mixture obtained from the red ball cap (Item 11). Based on the U.S. population, it is estimated that 1 in 5 individuals is a potential contributor to this profile. The DNA profile obtained from the bandana (Item 4) contained a mixture of DNA from at least two people. The major component is from a single male and the minor component is from at least one other person at trace levels. Henry Knox is eliminated as the source of the major component of this mixture. No interpretation is made of the trace component.

  16. STRBase • Everything you wanted to know about STRs • Online resource maintained by NIST • National Institute of Standards and Technology • http://www.cstl.nist.gov/strbase/ • Tour of STRbase • Further reading: John Butler’s “Fundamentals of Forensic DNA Typing” (Amazon $42)

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