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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 = 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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 Allele defined as the number of repeats at the
STR locus
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 E.g. GACA repeated 15 times in a row
# genotypes = (x 2 + x)/2
 E.g. ABO system # alleles = x = 3; # genotypes = 6
 E.g. vWA; # alleles = x = 14; # genotypes = 105!
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 Block is small enough for PCR amplification using primers flanking the repeated block
 Good for trace evidence and degraded DNA
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 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)
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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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 Primers 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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 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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Primer binding site mutations
Amplification artifacts
 Allelic drop out , allelic drop in, stutter
Electrophoretic artifacts
 Pull-up, dye blobs, and spikes
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Degraded DNA
 MiniSTR multiplex kits
Low-copy Number DNA (LCN)
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 < 100 pg of DNA
Mixtures
 Sexual assault cases
 Mixture interpretation
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 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
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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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Everything you wanted to know about STRs
Online resource maintained by NIST
 National Institute of Standards and Technology
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 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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