1. JS 190- Introduction to STRs I.Pre class activities a.Review Assignments and Schedules b.Exam 2 moved to Weds 11 April II. Learning Objectives a.Short Tandem Repeats 1. Biology of STRs 2. Fluorescence and Detection formats 3. Stutter 4. Statistics and Interpretation b.Other markers: mtDNA and Y STRs

2. Short Tandem Repeats: a subgroup of tandem repeats (Kuhl and Caskey 1993. 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.

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

5. Information on 13 CODIS STRs D18S51 18q21.3 AGAA L18333137-2743 Locus Name Chromosomal Location Repeat Motif ISFH format GenBank Accession Allele in GenBank Allele Range Number of Alleles Seen CSF1PO 5q33.3-34 TAGAX14720 126-1615 FGA 4q28 CTTT M649822115-51.269 TH01 11p15.5 TCAT D0026993-1420 TPOX 2p23-pter GAAT M68651116-1310 VWA 12p12-pter [TCTG][TCTA] M258581810-2428 D3S1358 3p [TCTG][TCTA] Not available--9-2020 D5S818 5q21-31 AGAT G08446117-1610 D7S820 7q11.21-22 GATA G08616126-1522 D8S1179 8 [TCTA][TCTG]G08710 128-1913 D13S317 13q22-31 TATC G09017135-1514 D16S539 16q24-qter GATA G07925115-1510 D21S1121q21 Complex [TCTA][TCTG] AP0004332924-3870

6. 520540560580 600 620640 WAVELENGTH (nm) 100 80 60 40 20 0 5-FAMJOENEDROX Laser excitation (488, 514.5 nm) Laser excitation (488, 514.5 nm) Normalized Fluorescent Intensity Fluorescent Emission Spectra for Dyes Filters collect light in narrow range Overlap is automatically calculated and subtracted using fluorescence “matrix” standards ABI 310 Filter Set F with color contributions between dyes

7. 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

8. Detection Formats Gel Electrophoresis Capillary Electrophoresis Microarrays (Nanogen) MALDITOF-MS (Sequenome)

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

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

11. 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

12. 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)

13. 1432VS CSF1PO TPOX Amelogenin THO1 vWA

14. Capillary Electrophoresis System Laser Inlet Buffer Capillary filled with polymer solution 5-20 kV -+ Outlet Buffer 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

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

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

17. 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. Electrical Current Smaller fragments Larger fragments

18. 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.

19. Laser - Camera 145 146 150 153 157

20. 145 Laser - Camera 146 150 153 157

21. Laser - Camera 146 150 153 157

22. Laser - Camera 150 153 157

23. Laser - Camera 153 157

24. STR Peaks - What do They Represent? 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. Larger allelic fragments Smaller allelic fragments

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

26. Comparison of Gels vs CE Gels –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

27. Heterozygous versus Homozygous in SINGLE SOURCE samples Locus 1Locus 2Locus 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. Heterozygous Homozygous

28. 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

29. STR Allele Frequencies Caucasians (N=427) Blacks (N=414) Hispanics (N=414) TH01 Marker * Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34 Number of repeats Frequency

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

31. FBI’s CODIS DNA Database Combined DNA Index System: http://www.fbi.gov/about-us/lab/codis/ndis-statistics 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 February 2012 –Total number of profiles: 10,560,300 –Total Forensic profiles: 417,200 –Total Hits: 173,500 –166,700Investigations Aided

32. 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.

33. 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

34. 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