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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 - PowerPoint PPT Presentation
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Forensic Biologyby Richard Li, with additions and edits by Ruth Ballard
Lecture 12: Autosomal STR DNA Profiling
Outline
STRs as DNA Markers Amplification by end-point PCR
AmpFlSTR Identifiler 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
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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)
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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!
Amplification by end-point PCRBlock is small enough for PCR
amplification using primers flanking the repeated block Good for trace evidence and degraded
DNA
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Amplification by end-point PCRSeveral 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)
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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
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Amplification by end-point PCRPrimers are tagged with fluorescent
dyes The primers get incorporated into the
amplicons, thereby labeling them
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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
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Amplification by end-point PCR
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
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Relative fluorescent units (rfu’s)
Time since injection = amplicon length
Factors Affecting Genotyping ResultsPrimer binding site mutationsAmplification artifacts
Allelic drop out , allelic drop in, stutterElectrophoretic artifacts
Pull-up, dye blobs, and spikes
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Genotyping of Challenging Forensic SamplesDegraded DNA
MiniSTR multiplex kitsLow-copy Number DNA (LCN)
< 100 pg of DNAMixtures
Sexual assault cases Mixture interpretation
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Interpretation of ResultsSWGDAM & 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
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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.
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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 STRbaseFurther reading: John Butler’s
“Fundamentals of Forensic DNA Typing” (Amazon $42)
Recommended