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

2. 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

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
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

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

10. 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

11. 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)

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

13. 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

14. 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

15. Interpretation of Results
SWGDAM & DNA Commission of the ISFG:
Inclusion (Match)
Calculate RMP
Sometimes challenged in Court (especially mixtures)
Exclusion
No calculation needed
Inconclusive
Multiple interpretations may be possible
Often challenged in Court

16. 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.

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