Lecture 12: Autosomal STR DNA Profiling. STRs as DNA Markers Amplification by end-point PCR AmpFlSTR Identifiler system Separation of amplicons.

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

reactions7

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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 Results

Primer 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 Samples

Degraded DNA MiniSTR multiplex kits

Low-copy Number DNA (LCN) < 100 pg of DNA

Mixtures Sexual assault cases Mixture interpretation

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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

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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)