Biology of STRs

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Biology of STRs

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Biology of STRs

• Stutter products
• Adenylated products
• Microvariants (Off-ladder alleles)
• Allele dropout and null alleles
• Mutations

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Stutter Products

• Stutter Product peaks are small peaks that are
  typically one repeat unit smaller than main peak.

Also, shadow band or DNA polymerase slippage
product
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Stutter Products

• Small peaks only a small percentage of the main
  allele.

Stutter peak is 4 bp less than main allele.
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Stutter Products

• What causes stutter products?

• Evidence somewhat unclear.
• Theory revolves around slipped-strand mispairing.

Taq
Primer
ACGATCGTTCGATCTATCTATCTAT
ACGATCGTAGCTAGATAGATAGATAGATAGATATCTAGG
Replication begins
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Stutter Products

• What causes stutter products?

• Evidence somewhat unclear.
• Theory revolves around slipped-strand mispairing.

Taq
Primer
ACGATCGTTCGATCTATCTATCTAT
ACGATCGTAGCTAGATAGATAGATAGATAGATATCTAGG
Replication pauses, temporarily unpaired
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Stutter Products

• What causes stutter products?

• Evidence somewhat unclear.
• Theory revolves around slipped-strand mispairing.

Taq
Primer
ACGATCGTTCGATCTATCTATCTAT
ACGATCGTAGCTAGATAGATAGATAGATATCTAGG
GA AT
One repeat forms non-base-paired loop.
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Stutter Products

• What causes stutter products?

• Evidence somewhat unclear.
• Theory revolves around slipped-strand mispairing.

Taq
Only 5 repeats
Primer
ACGATCGTTCGATCTATCTATCTATCTATAGATCC
ACGATCGTAGCTAGATAGATAGATAGATATCTAGG
GA AT
One repeat forms non-base-paired loop.
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Stutter Products

• Why is stutter important?

• Biological evidence may be a mixture of genotypes
  (gt1 contributor)
• Mixtures often have major and minor contributors.
• Size of minor contributor and stutter peaks are
  approximately the same (stutter lt15).

Stutter peaks vs. small real alleles
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Stutter Products

• Stutter peaks vs. small real alleles

Stutter is reported as a percentage (stutter
height / main peak height)
Peak Heights 100 4000 350 4000
Stutter
2.5
8.8
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Stutter Products

• Stutter peaks vs. small real alleles

Stutter can be compared to known stutter for
known primer sets.
5 at allele 6 of TH01
5 at allele 11 of D7S
probably not stutter
could be stutter
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Stutter Products

• What affects stutter?

• Size of repeat unit (di-, tri-nucleotides have
  more stutter)
• More common in simple repeats. Base pairing might
  not match up in complex repeats.
• Speed of polymerase (faster less time for pause
  less stutter)

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Stutter Products

• Solution to stutter products?

• STR analysis at other loci with reduced stutter
  (pentanucleotide repeats Penta A G)

• Use a faster polymerase

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Adenylation of PCR Products

• One trait of Taq polymerase is the addition of a
  nucleotide to the 3 end of a product

Taq
CATCGATAGATCCTGCTAGCTAGCC
Primer
ACGATCGTAGCTATCTAGGACGATCGATCGG
Two possibilities at end of replication 1. Taq
falls off (-A form)
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Adenylation of PCR Products

• One trait of Taq polymerase is the addition of a
  nucleotide to the 3 end of a product

CATCGATAGATCCTGCTAGCTAGCC
Primer
Taq
ACGATCGTAGCTATCTAGGACGATCGATCGG
Two possibilities at end of replication 1. Taq
falls off (-A form)
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Adenylation of PCR Products

• One trait of Taq polymerase is the addition of a
  nucleotide to the 3 end of a product

Taq
CATCGATAGATCCTGCTAGCTAGCCA
Primer
ACGATCGTAGCTATCTAGGACGATCGATCGG
Two possibilities at end of replication 2. Taq
adds A (most often),
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Adenylation of PCR Products

• One trait of Taq polymerase is the addition of a
  nucleotide to the 3 end of a product

Taq
CATCGATAGATCCTGCTAGCTAGCCA
Primer
ACGATCGTAGCTATCTAGGACGATCGATCGG
Two possibilities at end of replication 2. Taq
adds A (most often), then falls off (A)
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Adenylation of PCR Products
PCR conditions will favor one of the forms.
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Adenylation
Factors affecting adenylation

• Primer sequence

ATCCTGCTAGCTAGCCA
5 G favors complete adenylation
TAGGACGATCGATCGG

• Final incubation step of 60 C favors adenylation

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Adenylation
Effect of partial adenylation
Either form is fine, as long as all the PCR
products are in that form.
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Adenylation
Problems with adenylation

• Partial adenylation leads to double peaks,
  shoulders, wide peaks. Difficult to call.
• May be caused by too much DNA

10 ng
2 ng

• If sample is different from standard (allelic
  ladder)
• A form of 9.3 TH01 A form of 10 TH01
• For STR kits, all samples must be in same form.

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Microvarients or Off-Ladder

• Rare alleles may exist that are different from
  more common alleles.
• Sequence variation (point mutation)
• Insertions or deletions

Sequence variation will not affect STR alleles
dependant on size.
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Microvarients gt Insertions
Insertion of 1 bp
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Microvarients

• Nomenclature for microvarients
• Allele number followed by .x, where x is the
  number of bases of the incomplete repeat
• Example 9.3, 24.1, 23.2, etc.

• Other weird microvarients
• Alleles outside ladder
• Three peak pattern

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Allele Dropout or Null Alleles

• Mutations in the primer binding sites may result
  in an allele failing to amplify.

The genotype given by the kit will be different
than the actual genotype.
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Allele Dropout

• Example

An individual has a 6 and 8 allele at TH01 locus.
Normal
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Allele Dropout

• Example

An individual has a 6 and 8 allele at TH01 locus.
Normal
Primer
GCTATAGCTAGGTAC
CGATATCGATCCATGCTTA
Bing!
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Allele Dropout

• Example

An individual has a 6 and 8 allele at TH01 locus.
Primer
GCTATAGCTAGGTAC
and
CGATATCGATACATGCTTA
A mutation in the primer region.
Bong!
No Match
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Mutations

• Rarely, a mutation occurs and a childs genotype
  will not match one parent.
• Usually gain/loss of single repeat unit
• Mutation rare (1 in 1000)
• Mutations higher in more variable STRs.

Normally, one mutation will be caught due to
matches at other maternal/paternal loci.