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JS 115- Introduction to STRs

JS 115- Introduction to STRs. Pre class activities Review Assignments and Schedules Return and Review Exam 1 II. Learning Objectives (C5 ) Short Tandem Repeats 1. Biology of STRs 2. Fluorescence and Detection formats 3. Stutter 4. Statistics and Interpretation

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JS 115- Introduction to STRs

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  1. JS 115- Introduction to STRs • Pre class activities • Review Assignments and Schedules • Return and Review Exam 1 • II. Learning Objectives(C5) • Short Tandem Repeats • 1. Biology of STRs • 2. Fluorescence and Detection formats • 3. Stutter • 4. Statistics and Interpretation • 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.

  3. Fluorescent dye label AATG AATG AATG Short Tandem Repeats (STRs) 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

  4. Locus Name Chromosomal Location Repeat Motif ISFH format GenBank Accession Allele in GenBank Allele Range Number of Alleles Seen CSF1PO 5q33.3-34 TAGA X14720 12 6-16 15 FGA 4q28 CTTT M64982 21 15-51.2 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 7q11.21-22 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 D21S11 21q21 Complex [TCTA][TCTG] AP000433 29 24-38 70 Information on 13 CODIS STRs D18S51 18q21.3 AGAA L18333 13 7-27 43

  5. 5-FAM JOE NED ROX 100 80 60 Normalized Fluorescent Intensity 40 20 0 600 520 540 560 580 620 640 WAVELENGTH (nm) Laser excitation (488, 514.5 nm) Fluorescent Emission Spectra for DyesFilters collect light in narrow rangeOverlap is automatically calculated and subtracted using fluorescence “matrix” standards ABI 310 Filter Set F with color contributions between dyes

  6. 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 Multiplex PCR

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

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

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

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

  11. 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)

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

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

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

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

  16. Electrical Current 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 Smaller fragments

  17. 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.

  18. 157 153 150 146 Laser - Camera 145

  19. 157 153 150 Laser - Camera 146 145

  20. 157 153 Laser - Camera 150 146

  21. 157 Laser - Camera 153 150

  22. 157 Laser - Camera 153

  23. 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.

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

  25. 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 Comparison of Gels vs CE

  26. Heterozygous versus Homozygousin SINGLE SOURCE samples Locus 1 Heterozygous Locus 2 Heterozygous Locus 3 Homozygous 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.

  27. 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

  28. 45 40 35 30 Caucasians (N=427) 25 Frequency Blacks (N=414) 20 Hispanics (N=414) 15 10 5 0 6 7 8 9 9.3 10 Number of repeats STR Allele Frequencies TH01 Marker *Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

  29. 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

  30. FBI’s CODIS DNA Database Combined DNA Index System: http://www.fbi.gov/hq/lab/codis/index1.htm • 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 September 2003 • Total number of profiles: 1,472,150 • Total Forensic profiles: 64,523 • Total Convicted Offender Profiles: 1,407,627 • 9,842 Investigations Aided through September 2003

  31. 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.

  32. 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

  33. Summary 1 • Review of DNA Function and Structure • DeoxyriboNucleic Acid : blueprints of life 3 main functions: RIM • 1) Replication • 2) Information Storage • 3) Mutation for variation • Central Dogma information flow---------------> DNA------->RNA------>protein transcription translation • Function: • RIM- Pacific RIM- Replication-Information-Mutation • Information Storage- Phone Number analogy-Sequence • Structure: • PBS- The only station Sierra and Gabriel can watch • Asian Guys Can Teach: AGCT • DNA is where its AT • DNA velcro (David Letterman

  34. 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

  35. 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

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