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Unformatted text preview: AAFS 2006 Workshop (Butler and McCord) Advanced Topics in STR DNA Analysis February 20, 2006 http://www.cstl.nist.gov/biotech/strbase/training.htm 1 Advanced Topics in STR DNA Analysis AAFS 2006 Workshop #6 Seattle, WA February 20, 2006 Dr. John M. Butler Dr. Bruce R. McCord CE Troubleshooting [email protected] [email protected] Outline for This Section 1. Chemistry/molecular biology problems – stutter, -A, degradation, inhibition, low copy # 2. Sample and buffer problems – formamide, urea, water, salt concentration, free dye (“dye blobs”) 3. External factors – power supply, room temperature, cleanliness, voltage leaks 4. Instrument problems – optical system, capillary clogging, air bubbles, syringe leaks 5. Troubleshooting benchmarks/QC monitoring CE Troubleshooting 1. Chemistry/Molecular Biology Problems • PCR amplification issues • Adenylation • Stutter • Non Specific Amplification • Primer dimers • Pipetting small amounts • Degradation/Inhibition • Allele dropout • Over amplification • Ski slope effect • Mitigation Steps for inhibition Off-ladder alleles Four types 1 2 3 4 1. Spike 2.OL Allele 3. Free Dye 4. Noise Extract of Pistol Grip and Trigger Relatively low amount of amplified DNA? Blue Green Yellow Enhanced stutter or mixture? Truncated peaks give wrong ratios for peak stutter Why else is overloading bad? 1. raised baseline 2. non specific amplification 3. peak height ratios 4. -A Overloaded peaks will also show relatively high stutter Non specific amplification-A Stutter AAFS 2006 Workshop (Butler and McCord) Advanced Topics in STR DNA Analysis February 20, 2006 http://www.cstl.nist.gov/biotech/strbase/training.htm 2 Degradation and PCR Inhibition • Degradation affects larger alleles more, however there is no published study on the “threshold at which degradation is apparent”- The amplification efficiency of each set of alleles varies independently and differential amplification across loci can occur – Moretti, JFS 2001- Low quality formamide can mimic the degradation effect- Inhibition generally affects certain loci more than others and may or may not produce a slope effect- McCord, unpublished- There are several likely mechanisms for inhibition including DNA aggregation, Protein-DNA binding, chelation of Mg, interference with primer binding, etc. Non-degraded Positive Control 20 pg/μL (0.250 ng/12.5 μL) Degraded Bone Sample 20 pg/μL(0.250 ng/ 12.5 μL) DNA Degradation Degraded DNA and Amplification Allele drop- out Allele drop- out Oversaturation “Pull up”-A peak Enhanced stutter With degraded DNA two injections may be necessary to keep data on-scale Non-DNA Contamination/Inhibition • Anything that is water soluble may co-extract with DNA unless a capture technique is used....
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- DNA, STR DNA Analysis, Dr. John M. Butler Dr. Bruce R. McCord