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STR Interpretation 1

STR Interpretation 1 - Allelic Ladder STR Analysis is...

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Allelic Ladder STR Analysis is Performed by Comparing Size of Questioned Peak to Allelic Ladders Variant allele Outline g122 What are STRs and how are they amplified g122 STR applications in forensics Capillary electrophoresis Analysis of DNA data using Genemapper g122 STR analysis and interpretation issues Case examples g122 Additional STR applications Y Chromosome STRs Practical Considerations Regarding DNA Data Analysis g122 Stutter g122 -A/+A g122 Amplification locus imbalance g122 Pull-up g122 Degraded template g122 Is it a real peak? Stochastic fluctuation, spikes, rfu thresholds, and minor peaks g122 Mixture interpretation g122 Primer binding microvariants Stutter Stutter – a reproducible characteristic of Taq activity. …. not an “artifact”. Stutter is the result of repeat slippage during the primer extension step of PCR. Mechanism: Taq polymerase can stall and fall off during DNA synthesis. The extended stand breathes apart and can reanneal out of register. The most common result is a minor peak at N-4 base pairs. RFU 100 104 Size (bp) Stutter is common. Observed at most STR loci used in forensic testing. Peaks are identified as stutter based on validation studies: testing known samples (empirically define stutter percentage or “stutter cut-offs”). --by the kit manufacturer --by the user laboratory Stutter Characteristics General trends: g57 Larger amplification products have higher stutter percentage. -Locus to locus: generally more stutter is observed at larger loci. -Within a locus: larger alleles usually have more stutter.
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Case Sample with Stutter Real peaks Below Stutter Threshold 5- fold more product 10% 10% Actual % stutter is the same Threshold Threshold of detection is exceeded is exceeded for main peak …but not for stutter peak 22% 10% Percent Stutter is Falsely Augmented
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