Chapter 6 Biology of STRs: Stutter Products, Non-template Addition, Microvariants, Null Alleles, and Mutation Rates ©2002 Academic Press.

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Presentation transcript:

Chapter 6 Biology of STRs: Stutter Products, Non-template Addition, Microvariants, Null Alleles, and Mutation Rates ©2002 Academic Press

Biological “Artifacts” of STR Markers Stutter Products Non-template nucleotide addition Microvariants Null alleles Mutations ©2002 Academic Press

Stutter Products Peaks that show up primarily one repeat less than the true allele as a result of strand slippage during DNA synthesis Stutter is less pronounced with larger repeat unit sizes (dinucleotides > tri- > tetra- > penta-) Longer repeat regions generate more stutter Each successive stutter product is less intense (allele > repeat-1 > repeat-2) Stutter peaks make mixture analysis more difficult ©2002 Academic Press

STR Alleles with Stutter Products DNA Size (bp) D8S1179 D18S51 D21S11 Allele Relative Fluorescence Units Stutter Product 6.3% 6.2% 5.4% ©2002 Academic Press

Higher Levels of DNA Lead to Incomplete Adenylation D3S1358 VWA FGA -A +A 10 ng template (overloaded) 2 ng template (suggested level) DNA Size (bp) Relative Fluorescence (RFUs) off-scale ©2002 Academic Press

Non-template Addition 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 A ©2002 Academic Press

Impact of the 5’ nucleotide on Non-Template Addition 5’-CCAAG… 5’-ACAAG… Last Base for Primer Opposite Dye Label ©2002 Academic Press

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 ©2002 Academic Press

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 Can cause PCR failure due to polymorphism in the primer site -- “null alleles” ©2002 Academic Press

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 28.1 ©2002 Academic Press

Three-Peak Pattern at D18S51 AMEL D8S1179 D21S11 D18S51 ©2002 Academic Press

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 ©2002 Academic Press

Impact of DNA Sequence Variation in the PCR Primer Binding Site * 8 6 Allele 6 amplicon has “dropped out” Imbalance in allele peak heights Heterozygous alleles are well balanced ©2002 Academic Press

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 ©2002 Academic Press

Mutation Observed in Family Trio 14,18 13,17 15,17 14,18 15,18 15,17 Normal Transmission of Alleles (No Mutation) Paternal Mutation ©2002 Academic Press

Measured Mutation Rates Apparent Mutations Observed at the 13 CODIS STR Loci in the Course of Paternity Testing* Measured Mutation Rates STR 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) 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) D5S818 22/60907 (0.04) 194/130833 (0.15) 3/74922 (<0.01) 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) D13S317 33/59500 (0.06) 106/69598 (0.15) 52/62344 (0.08) 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) D21S11 12/6754 (0.18) 17/6980 (0.24) 1/203 (0.49) http://www.cstl.nist.gov/biotech/strbase/mutation.htm *Data used with permission from American Association of Blood Banks (AABB) 1999 Annual Report.