JS 115- Introduction to STRs- Continued

I. Pre class activitiesa. Review Assignments and Schedules

1. Assignment- Read Chapters 6 and 7 Butler, Ch 7 Rudin2. 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 revisited2. Biology of STRs- Define-

BalanceStutter ProductsNon-template AdditionMicrovariantsNull AllelesMutation Rates

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

520 540 560 580 600 620 640WAVELENGTH (nm)

100

80

60

40

20

0

5-FAM JOE NED ROX

Laser excitation(488, 514.5 nm)

Normalized Fluorescent Intensity

Fluorescent Emission Spectra for DyesFilters 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

Dye overlap shown in raw dataAutomatically subtracted in processed data (BGYR)

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.

Trinucleotide Repeats Implicated in Human Diseases

Sutherland and Richards. 1994. Dynamic Mutations American Scientist

82:157 Disease Chrom Parental

SexBias of severe form

REPEAT SEQ

>66%GC

# Normal # Pre- mutation

# full mutation

Fragile X syndrome

X maternal CCG 6-50 50-230 230-2000

spino- bulbar

muscular atrophy

(Kennedy disease)

X ? AGC 11-31 40-62

myotonic dystrophy

19 maternal AGC 5-35 50-80 80-2000

Huntington

Disease

4 paternal AGC 9-37 30-38 37-121

spinocere-bellar

ataxia type 1

6 paternal ?

AGC 25-36 43-81

FRAXE X ? CCG 6-25 25-200 200++ dentatorub

ral and palliduluysian atrophy

12 (mainly)

paternal AGC

7-23 49-754

Trinucleotide repeat expansion for Fragile X

syndrome in the FMR-1 gene 

Copies of CGG 'Phenotype' CGG 6-54 NormalCGGCGG 50-200 Normal Transmitting MaleCGGCGG 50-200 DaughterCGGCGGCGG 200-3000 Affected Individual

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.

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

Multiplexing provides powerful discrimination

• # Loci Most Common Reference• 3 1/500 individuals Edwards

Edwards et al. 1994. AJHG 55:175

• 6 1/200,000 AJHG 49:746• 9 1/300,000,000 (nineplex)Walsh

• 13 (CODIS loci)• 1/100,000,000,000,000 Walsh 98 JFS

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

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.

Balance within and among loci

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 oC)• 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.)

AA

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+.

D3S1358 VWAFGA

-A

+A10 ng template (overloaded)

2 ng template (suggested level)

DNA Size (bp)

Relative Fluorescence (RFUs)off-scale

Higher Levels of DNA Lead to Incomplete Adenylation

+A +A

-A+A+A

-A 5’-CCAAG…

5’-ACAAG…

Last Base for Primer Opposite Dye Label

Impact of the 5’ nucleotide on Non-Template Addition

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

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.

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-)

STR Alleles with Stutter Products

D21S11 D18S51

D8S1179

DNA Size (bp)

Stutter Product

6.3% 6.2% 5.4%

Allele

Rel

ativ

e Fl

uore

scen

ce U

nits

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

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

28.1

Detection of a Microvariant Allele at the STR locus FGA

1 = S25-L25 = 244.34 - 244.46 = -0.12 bp

2 = SOL - L28 = 257.51-256.64 = +0.87 bp

c = |1 -2| = |-0.12-0.87| = 0.99 bp

Three-Peak Pattern at D18S51

AMELD8S1179 D21S11

D18S51

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

*

*8

86

6 8

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

Mutation Observed in Family TrioMutations 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

15,17 14,18

13,17

15,17

Normal Transmission of Alleles (No Mutation)

Paternal Mutation

Measured Mutation RatesSTR Locus Maternal Meioses (%) Paternal Meioses (%) Null Alleles (%) Multi-Banded (%)

CSF1PO 14/47843 (0.03) 311/243124 (0.13) 2/42020 (<0.01) None reported

FGA 7/8253 (0.01) 555/189973 (0.29) 2/1104 (0.18) None reported

TH01 5/42100 (0.01) 12/74426 (0.02) 2/7983 (0.03) 0/2646 (<0.040)

TPOX 2/28766 (0.01) 10/45374 (0.02) 11/43704 (0.03) 13/42020 (0.03)

VWA 20/58839 (0.03) 851/250131 (0.34) 7/42220 (0.02) 1/6581 (0.02)

D3S1358 0/4889 (<0.02) 9/8029(0.11) None reported None reported

D5S818 22/60907 (0.04) 194/130833 (0.15) 3/74922 (<0.01) None reported

D7S820 14/50827 (0.03) 193/131880 (0.15) 1/42020 (<0.01) 1/406 (0.25)

D8S1179 5/6672 (0.07) 29/10952 (0.26) None reported None reported

D13S317 33/59500 (0.06) 106/69598 (0.15) 52/62344 (0.08) None reported

D16S539 12/42648 (0.03) 40/48760 (0.08) 3/52959 (<0.01) 0/1165 (<0.09)

D18S51 8/8827 (0.09) 29/9567 (0.30) None reported None reported

D21S11 12/6754 (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.

Review of STRsIntro 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