Download - STR lecture part 2 [PPT]
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