1. JS 115- Introduction to STRs- Continued I.Pre class activities a.Review Assignments and Schedules 1.Assignment- Read Chapters 6 and 7 Butler, Ch 7 Rudin 2.Optional assignment- Read Scientific American article on microsattellites- See Lee for copy- 500 word summary with 3 Q ad 3 A II. Learning Objectives (C6 Butler ) a.Short Tandem Repeats 1. CE artifacts and Fluorescent Dye multiplexing revisited 2. Biology of STRs- Define- Balance Stutter Products Non-template Addition Microvariants Null Alleles Mutation Rates

2. CE artifact:Spikes in formamide blank- 4 colors (S. Myers-CA DOJ DNA) CE artifact: Spikes in one color- no stutter, pull down These are not real DNA peaks

3. 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

4. Dye overlap shown in raw data Automatically subtracted in processed data (BGYR)

5. 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.

6. Trinucleotide Repeats Implicated in Human Diseases Sutherland and Richards. 1994. Dynamic Mutations American Scientist 82:157

7. Trinucleotide repeat expansion for Fragile X syndrome in the FMR-1 gene Copies of CGG'Phenotype' CGG6-54 Normal CGGCGG50-200 Normal Transmitting Male CGGCGG50-200 Daughter CGGCGGCGG200-3000Affected Individual

8. Advantages of STRs in Forensics All of the above and more! Common, polymorphic, stably inherited, well studied- discrete sizes Small size and size range- Useful on highly degraded samples Small size range- Less prone to preferential amplification of the smaller allele Multiple STRs provide powerful discrimination Abundance permits choice of STRs with non overlapping size ranges. Even for those with overlapping sizes, use of different color fluorescently tagged primers permit rapid automated analysis.

9. Small size and small size range permit typing of highly degraded samples 73 pathological samples exposed to high temperature, incineration, explosion and chemical insult. Waco disaster: All four loci success 63%, at least 1 locus 83% VWFA31, THO1, F13A01, FES/FPS Whitaker et al. Biotechniques 19:670

10. Multiplexing provides powerful discrimination # LociMost CommonReference 31/500 individualsEdwards Edwards et al. 1994. AJHG 55:175 61/200,000AJHG 49:746 91/300,000,000 (nineplex)Walsh 13 (CODIS loci) 1/100,000,000,000,000Walsh 98 JFS

11. Biological Issues and “Artifacts” of STR Markers Balance of results Non-template nucleotide addition- aka. N+1, aka. 'split peaks', aka. incomplete extension Stutter Products- aka. Repeat slippage Microvariants – aka. Deletions Null alleles- primer binding site mutations Mutations

12. Balance of results among loci In multiplex PCR reactions, some loci may amplify more efficiently than others. Ideally, individual loci in a multiplex should not differ in signal intensity by more than about 10-20%, thereby insuring that mixtures can, in most circumstances, be easily sorted out. A multiplex which may exhibit perfect signal balance with pristine DNA may, however, show preferential amplification with "forensic type" samples, presumably due to the alteration of the reaction environment by the addition of contaminants which co-purify with the DNA.

13. Balance within and among loci

14. Non template directed nucleotide addition to blunt ends (aka. N+1, split peaks, incomplete extension) Taq polymerase will often add an extra nucleotide to the end of a PCR product; most often an “A” Dependent on 5’-end of the reverse primer Can be enhanced with extension soak at the end of the PCR cycle (e.g., 15-45 min @ 60 or 72 o C) Can be reduced with new polymerase Best if there is NOT a mixture of “+/- A” peaks (Clark,J. NAR 16:9677, Hu. 1993. DNA and Cell Biol. 12:763.) A A

15. Non template directed nucleotide addition to blunt ends A property of the Taq (and other DNA polymerases), not specific to STRs where an extra nucleotide is added to the 3'OH end of blunt ended double stranded DNA Problem when it is not 100% because peaks (bands) are split (two peaks for the same product, one base pair apart). It is sequence specific, so not all loci will exhibit, and is effected by rxn conditions (eg Mg2+). For STRs resolved by adding an extension at the end of thermal cycling. The extension to favor nt+ is currently done at 60C for 30 minutes. The lower temp is used to reduce 'breathing' between the template and extending strand. The choice of primer sequence can influence the amount of nt+.

16. D3S1358 VWA FGA -A +A 10 ng template (overloaded) 2 ng template (suggested level) DNA Size (bp) Relative Fluorescence (RFUs) off-scale Higher Levels of DNA Lead to Incomplete Adenylation

17. +A -A +A -A 5’-CCAAG… 5’-ACAAG… Last Base for Primer Opposite Dye Label Impact of the 5’ nucleotide on Non-Template Addition

18. Stutter or Repeat Slippage Definition: Peaks that show up primarily one repeat less than the true allele as a result of strand slippage during DNA synthesis (-n where n=1 repeat = 4bp). Faint peaks or bands which are sized as true allele -n, -2n, -3n…). Each successive stutter product is less intense (allele > repeat-n > repeat- 2n>repeat-3n) All DNA polymerases seem to do it (in fact this phenomena occurs in genetic diseases resulting from repeat expansion). In most forensic STR systems we usually only see the repeat-n stutter product

19. Stutter as it correlates to allele size (eg number of repeats) Levels of repeat slippage vary for different loci and even for the different alleles of a particular locus. Amount of repeat slippage appears to be greater in larger alleles with more repeats and less in those that are smaller. Longer repeat regions generate more stutter. That is, a 20 repeat allele will generally have more stutter than a 10 repeat allele Amount of slippage for a given sized allele appeared to be quite reproducible.

20. Stutter as it correlates to unit size (eg the number of bases in a single repeat) Stutter is not as bad with larger repeat unit sizes. Very bad with small size- di-nucleotide repeats. Not as bad with larger size - tetra and penta nucleotide repeats (dinucleotides > tri- > tetra- > penta-)

21. STR Alleles with Stutter Products D21S11 D18S51 D8S1179 DNA Size (bp) Stutter Product 6.3%6.2% 5.4% Allele Relative Fluorescence Units

22. Microvariant Alleles Not all alleles have full length repeat units Alleles with partial repeat units are designated by the number of full repeats and then a decimal point followed by the number of bases in the partial repeat Example: TH01 9.3 allele (AATG) 6 (-ATG)(AATG) 3

23. Microvariants Defined as alleles that are not exact multiples of the basic repeat motif or sequence variants of the repeat motif or both May exist as insertion, deletion, or base change Sequence variation can occur within repeat, in the flanking region, or in a primer binding site

24. 28.1 Detection of a Microvariant Allele at the STR locus FGA  1 = S 25 -L 25 = 244.34 - 244.46 = -0.12 bp  2 = S OL - L 28 = 257.51-256.64 = +0.87 bp c = |  1 -  2 | = |-0.12-0.87| = 0.99 bp

25. Three-Peak Pattern at D18S51 AMEL D8S1179D21S11 D18S51

26. Null Alleles Allele is present in the DNA sample but fails to be amplified due to a nucleotide change in a primer binding site Allele dropout is a problem because a heterozygous sample appears falsely as a homozygote Two PCR primer sets can yield different results on samples originating from the same source This phenomenon impacts DNA databases Large concordance studies are typically performed prior to use of new STR kits

27. * * 8 8 6 68 Allele 6 amplicon has “dropped out” Imbalance in allele peak heights Heterozygous alleles are well balanced Impact of DNA Sequence Variation in the PCR Primer Binding Site

28. Mutation Observed in Family Trio Mutations may be detected in children Occur at approx 0.1-0.3% at each STR locus and appear to show a paternal bias- Dads STR change more frequently than Moms 14,18 15,18 15,1714,18 13,17 15,17 Normal Transmission of Alleles (No Mutation) Paternal Mutation

29. Measured Mutation Rates Apparent Mutations Observed at the 13 CODIS STR Loci in the Course of Paternity Testing* STR LocusMaternal Meioses (%)Paternal Meioses (%)Null Alleles (%)Multi-Banded (%) CSF1PO14/47843 (0.03)311/243124 (0.13)2/42020 (<0.01)None reported FGA7/8253 (0.01)555/189973 (0.29)2/1104 (0.18)None reported TH015/42100 (0.01)12/74426 (0.02)2/7983 (0.03)0/2646 (<0.040) TPOX2/28766 (0.01)10/45374 (0.02)11/43704 (0.03)13/42020 (0.03) VWA20/58839 (0.03)851/250131 (0.34)7/42220 (0.02)1/6581 (0.02) D3S13580/4889 (<0.02)9/8029(0.11)None reported D5S81822/60907 (0.04)194/130833 (0.15)3/74922 (<0.01)None reported D7S82014/50827 (0.03)193/131880 (0.15)1/42020 (<0.01)1/406 (0.25) D8S11795/6672 (0.07)29/10952 (0.26)None reported D13S31733/59500 (0.06)106/69598 (0.15)52/62344 (0.08)None reported D16S53912/42648 (0.03)40/48760 (0.08)3/52959 (<0.01)0/1165 (<0.09) D18S518/8827 (0.09)29/9567 (0.30)None reported D21S1112/6754 (0.18)17/6980 (0.24)1/203 (0.49)None reported http://www.cstl.nist.gov/biotech/strbase/mutation.htm *Data used with permission from American Association of Blood Banks (AABB) 1999 Annual Report.

30. 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 –Results are interpreted by reproducibility, size of the resulting fragment, spectral properties, stutter, and size of peak (balance within and among loci). –Multiplexing STR loci provide powerful discrimination