1. JS 196e- Introduction to STRs
Pre class activities
Quiz
Review Assignments and Schedules
II. Learning Objectives (C5)
Short Tandem Repeats
1. Biology of STRs
2. Fluorescence and Detection formats
3. Stutter
4. Statistics and Interpretation

2. Announcements and Assignments
Read Butler C5,6&7/ InmanC6-C7
Read posted article by Moxon and Wills – Microsatellites: Agents of Evolution Sci Amer. 280: 94 – Posted on my website
Read - Butler, J.M. (2006) Genetics and genomics of core STR loci used in human identity testing. J. Forensic Sci. 51(2): : Posted on my website
Visit- Web Link-
Additional optional readings
Budowle et al JFS 46: (CODIS STR Pop)
Walsh et al NAR. 24: , Levinson et al Mol Biol Evol. 4: , Brinkmann Am J Hum Genet 62:1408, Henke et al. Am J Hum Genet 64:1473.
Next time- Run Gels and Bruce Wiley 300

3. Short Tandem Repeats: a subgroup of tandem repeats (Kuhl and Caskey Curr. Opin. in Genet. Dev. 3:404)
Head to tail arrangements of sequence units (4bp),
Common in genomes (thousands distributed)
Polymorphic: vary in length by no. of and/or by content of repeats.
Stably inherited on a human time scale (for most)
Well studied b/c others are implicated in Human Diseases and therefore the subject of clinical studies.

5. Short Tandem Repeats (STRs)
Fluorescent dye label
AATG
AATG
7 repeats
8 repeats
the repeat region is variable between samples while the flanking regions where PCR primers bind are constant
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another
Primer positions define PCR product size

6. Information on 13 CODIS STRs
Locus Name
Chromosomal Location
Repeat Motif
ISFH format
GenBank Accession
Allele in GenBank
Allele Range
Number of Alleles Seen
CSF1PO 5q
TAGA
X14720 12
6-16 15
FGA
4q28
CTTT
M64982 21 69
TH01
11p15.5
TCAT
D00269 9
3-14 20
TPOX
2p23-pter
GAAT
M68651 11
6-13 10
VWA
12p12-pter
[TCTG][TCTA]
M25858 18
10-24 28
D3S1358 3p
[TCTG][TCTA]
Not available --
9-20 20
D5S818
5q21-31
AGAT
G08446 11
7-16 10
D7S820 7q
GATA
G08616 12
6-15 22
D8S1179 8
[TCTA][TCTG]
G08710 12
8-19 13
D13S317
13q22-31
TATC
G09017 13
5-15 14
D16S539
16q24-qter
GATA
G07925 11
5-15 10
D18S51
18q21.3
AGAA
L18333 13
7-27 43
D21S11
21q21
Complex [TCTA][TCTG]
AP000433 29
24-38 70

7. Normalized Fluorescent Intensity
Fluorescent Emission Spectra for Dyes Filters collect light in narrow range Overlap is automatically calculated and subtracted using fluorescence “matrix” standards
520
540
560
580
600
620
640
WAVELENGTH (nm)
100 80 60 40 20
5-FAM
JOE
NED
ROX
Laser excitation
(488, nm)
Normalized Fluorescent Intensity
ABI 310 Filter Set F with color contributions between dyes

8. Multiplex PCR 15 Markers Can Be amplified at once
Sensitivities to levels less than 1 ng of DNA
Ability to Handle Mixtures and Degraded Samples
Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges

9. Detection Formats Gel Electrophoresis Capillary Electrophoresis
Microarrays (Nanogen)
MALDITOF-MS (Sequenome)
Microcapillary Arrays (R. Matthies-Microchip Biotechnologies- Blazej et al PNAS 103:

10. Gel Electrophoresis System-
Voltage
Gel
Loading well +
anode
cathode
Side view
Top view
Gel lanes
DNA bands
Buffer

11. - + Separation of DNA sequence length amplified products Larger
fragments
Smaller
fragments +

12. FMBIO II Detection of STR Alleles
DNA samples are loaded onto a polyacrylamide gel
STR alleles separate during electrophoresis through the gel
Sample Separation
Sample Detection (Post-Electrophoresis)
505 nm scan to detect fluorescein-labels
585 nm scan to detect TMR-labels

13. Example of STR test result
15 different STR loci may be typed on a single gel
Scanned using a laser
and filters to assist in detecting different colors (fluor tags)

14. 1 2 3 4 V S
CSF1PO
TPOX
Amelogenin
THO1
vWA

15. Capillary Electrophoresis System
Laser
Inlet
Buffer
Capillary filled with polymer solution
5-20 kV - +
Outlet
Sample tray
Detection window
(cathode)
(anode)
Data Acquisition
Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession

16. Principles of CE Sample Separation and Detection
Labeled DNA fragments (PCR products)
Principles of CE Sample Separation and Detection
Capillary or Gel Lane
Size Separation
Sample Detection
CCD Panel
Ar+ LASER (488 nm)
Color
Separation
Detection region
ABI Prism spectrograph
Fluorescence

17. Results are interpreted and printed
Electropherogram: ABI Prism 310 Genetic Analyzer

18. STR Peaks - What do They Represent?
Going back to the gel electrophoresis, large PCR fragments travel slower than small PCR fragments as electricity is applied.
Larger
fragments
Electrical Current
Smaller
fragments

19. What STR Peaks Show
By the same token, smaller PCR fragments migrate through the capillary tube faster and thus are detected before the larger (slower) PCR fragments.

20. 157
153
150
146
145
Laser - Camera

21. 157
153
150
146
Laser - Camera
145

22. 157
153
150
Laser - Camera
146

23. 157
153
Laser - Camera
150

24. 157
Laser - Camera
153

25. STR Peaks - What do They Represent?
Smaller allelic
fragments
Larger allelic
fragments
NOTE: in an electropherogram,
-smaller DNA fragments (bottom of traditional gel) are on the left
- the larger fragments (top of the gel) are on the right.

26. STR Peaks - What do They Represent?
The area under the peak is directly proportional to the intensity of the signal.

27. Comparison of Gels vs CE
Advantages
Fewer artifacts
Generally less expensive
Less sensitive to ambient temperature
Disadvantages
Not fully automated
Need to pour and load gels
Cannot easily reinject a sample CE
Advantages
Real time detection
Better resolution of fragments and microvariants
Fuly automated- no gel pouring or loading
Can reinject samples
Majority of crime labs are using CE
Disadvantages
Generally more artifacts
More expensive
Temperature sensitive

28. Heterozygous versus Homozygous in SINGLE SOURCE samples
Locus 1
Locus 2
Heterozygous
Homozygous
Locus 3
At each locus there are either one or two peaks. Two peaks at a locus site are called heterozygous while one peak is called homozygous.

29. STR - Mixture and Stutter
Stutter is observed as a minor allele appearing one repeat unit smaller than the major STR allele. Some STR loci are more prone to stutter than others.
Stutter becomes an issue in putative mixed samples where a decision must be made whether a band is due to stutter or from another DNA source.
General Rule » Do stutter validation studies

30. STR Allele Frequencies5 10 15 20 25 30 35 40 45 6 7 8 9
9.3
TH01 Marker
Number of repeats
Frequency
Caucasians (N=427)
Blacks (N=414)
Hispanics (N=414)
*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

31. Probability Analysis - The Product Rule
1 in 10
Allele A has a frequency in a population of 1/10.
1 in 20
Allele B has a frequency in a population of 1/20.
1 in 5
Allele C has a frequency in a population of 1/5.
If all three alleles match in two samples then
1/10 x 1/20 x 1/5 = 1/1000

32. FBI’s CODIS DNA Database
Combined DNA Index System:
Used for linking serial crimes and unsolved cases with repeat offenders
Launched October 1998
Links all 50 states
Requires >4 RFLP markers
and/or 13 core STR markers
As of Jan 2007
Total number of profiles: 4,274,700
Total Forensic profiles: 163,689
Total Convicted Offender profiles: 4,111,011
44,567 Investigations Aided in 49 States and 2 Federal Laboratories.

33. Why mtDNA SNPs?
Well characterized and studied (population, evolutionary, medical and forensic studies)
Uniparental maternal inheritance missing persons-mat. lineage ref smpls
Relatively small size (16kb) and high copy number – good on low quantity/quality samples (hair, bone, teeth- ancient/degraded)-(Think Peterson case)
Implicated in maternally inherited diseases : diabetes, deafness, hypertrophic cardiomyopathy and myopathy
Analysis by DNA sequencing- more complex than STR analysis
mtDNA - many mitotypes are only found 1X. Some use counting method for statistics. Commonly found mitotypes are as frequent as 1 in 10.

34. Why Y?
Applications
Forensic investigations (98% of violent crime by men)
Biodefense- Male terrorist profiling
Genealogical and Evolutionary studies
Advantages to Human Identity Testing
Male component isolated without differential extraction
Paternal lineages
Some cases with no spermatazoa- use Y STRs
Assess number of male donors/contributors
Same analysis as autosomal STRs
Challenges
Y STR kits not as abundant- now 12plexes available in 2003
Some Y Haplogroups are common
Population specific haplotying needed for new markers

35. Review of PCR Review of PCR
PCR is repeated rounds of template directed, DNA replication
dNTPs added to 3’OH of a primer
Components are template, primers, dNTPs, Mg++ and taq polymerase.
Contamination prevention
separation of pre and post PCR areas, use of dedicated equipment, aerosol pipette tips and controls, process one sample at a time, separate reference samples from evidence, avoid splashing, wear protective gear and reagent prep care.
PCR is useful on degraded DNA. Due to specificity of primers, will not amplify non human DNA
Pitfalls- inhibitors, primer binding site mutations (rare), contamination

36. Review of STRs Intro to STRs
Head to tail arrangements 4 bp repeat units
Polymorphic, Common, Stably Inherited, Implicated in Diseases
Advantages- Discrete, Small- less prone to PA, Useful on highly degraded DNA, Ability to Multiplex , Provide powerful discrimination.
STR biological artifacts- stutter, adenylation, microvariants, null alleles, mutations
Multiplexing STR loci provide powerful discrimination