JS 196e- Introduction to STRs

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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

Announcements and Assignments Read Butler C5,6&7/ InmanC6-C7 Read posted article by Moxon and Wills – Microsatellites: Agents of Evolution 1999. 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): 253-265: Posted on my website Visit- Web Link- www.cstl.nist.gov/biotech/strbase Additional optional readings Budowle et al. 2001. JFS 46:453-489 (CODIS STR Pop) Walsh et al. 1996 NAR. 24:2807-2812, Levinson et al. 1987. Mol Biol Evol. 4:203-221, Brinkmann. 1998 Am J Hum Genet 62:1408, Henke et al. Am J Hum Genet 64:1473. Next time- Run Gels 130-245 and Bruce Wiley 300

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.

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

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   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 D18S51   18q21.3 AGAA L18333 13 7-27 43 D21S11 21q21 Complex [TCTA][TCTG] AP000433 29 24-38 70

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, 514.5 nm) Normalized Fluorescent Intensity ABI 310 Filter Set F with color contributions between dyes

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

Detection Formats Gel Electrophoresis Capillary Electrophoresis Microarrays (Nanogen) MALDITOF-MS (Sequenome) Microcapillary Arrays (R. Matthies-Microchip Biotechnologies- Blazej et al. 2006. PNAS 103:7240-7245

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

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

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

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)

1 2 3 4 V S CSF1PO TPOX Amelogenin THO1 vWA

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

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

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

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

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.

157 153 150 146 145 Laser - Camera

157 153 150 146 Laser - Camera 145

157 153 150 Laser - Camera 146

157 153 Laser - Camera 150

157 Laser - Camera 153

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.

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

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

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.

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

STR Allele Frequencies 5 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

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

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 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.

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.

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

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

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