JS 115 Introduction to STRs Continued

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JS 115- Introduction to STRs- Continued

• Pre class activities
• Review Assignments and Schedules
• Assignment- Read Chapters 6 and 7 Butler, Ch 7
  Rudin
• 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)
• 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

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CE artifactSpikes in formamide blank- 4 colors
(S. Myers-CA DOJ DNA)
These are not real DNA peaks

CE artifact Spikes in one color- no stutter,
pull down
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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
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Dye overlap shown in raw dataAutomatically
subtracted in processed data (BGYR)
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Short Tandem Repeats a subgroup of tandem
repeats (Kuhl and Caskey 1993. Curr. Opin. in
Genet. Dev. 3404)

• 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.

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Trinucleotide Repeats Implicated in Human
Diseases Sutherland and Richards. 1994. Dynamic
Mutations American Scientist 82157
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Trinucleotide repeat expansion for Fragile X
syndrome in the FMR-1 gene
  Copies of CGG 'Phenotype'   CGG 6-54
Normal CGGCGG 50-200 Normal Transmitting
Male CGGCGG 50-200 Daughter CGGCGGCGG 200-3000
Affected Individual
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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.

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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 19670

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Multiplexing provides powerful discrimination

• Loci Most Common Reference
• 3 1/500 individuals Edwards
• Edwards et al. 1994. AJHG 55175
• 6 1/200,000 AJHG 49746
• 9 1/300,000,000 (nineplex)Walsh
• 13 (CODIS loci)
• 1/100,000,000,000,000 Walsh 98 JFS

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Biological Issues and Artifacts of STR Markers

• Balance of results
• Non-template nucleotide addition- aka. N1, aka.
  'split peaks', aka. incomplete extension
• Stutter Products- aka. Repeat slippage
• Microvariants aka. Deletions
• Null alleles- primer binding site mutations
• Mutations

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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.

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Balance within and among loci
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Non template directed nucleotide addition to
blunt ends (aka. N1, 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 _at_ 60 or 72 oC)
• Can be reduced with new polymerase
• Best if there is NOT a mixture of /- A peaks
• (Clark,J. NAR 169677, Hu. 1993. DNA and Cell
  Biol. 12763.)

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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.

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Higher Levels of DNA Lead to Incomplete
Adenylation
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Impact of the 5 nucleotide on Non-Template
Addition
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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
  n1 repeat 4bp).
• Faint peaks or bands which are sized as true
  allele -n, -2n, -3n). Each successive stutter
  product is less intense (allele gt repeat-n gt
  repeat-2ngtrepeat-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

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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.

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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 gt tri- gt tetra- gt penta-)

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STR Alleles with Stutter Products
DNA Size (bp)
D8S1179
D18S51
D21S11
Allele
Relative Fluorescence Units
Stutter Product
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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

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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

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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
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Three-Peak Pattern at D18S51
AMEL
D8S1179
D21S11
D18S51
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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

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Impact of DNA Sequence Variation in the PCR
Primer Binding Site
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Mutation Observed in Family TrioMutations may be
detected in childrenOccur at approx 0.1-0.3 at
each STR locus and appear to show a paternal
bias- Dads STR change more frequently than Moms
Normal Transmission of Alleles (No Mutation)
Paternal Mutation
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Measured Mutation Rates
Apparent Mutations Observed at the 13 CODIS STR
Loci in the Course of Paternity Testing
http//www.cstl.nist.gov/biotech/strbase/mutation.
htm Data used with permission from American
Association of Blood Banks (AABB) 1999 Annual
Report.
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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