STR片段分析相关知识.pptVIP

* * * 1.Taq DNA Polymerase has extended through 4 repeat units 2.Taq has fallen off allowing the two strands to breathe apart. 3.When the two strands re-anneal the template (bottom) strand has looped out and the extending strand aligns out-of-register by one repeat unit. 4.The newly completed strand contains only 7 repeat units, while the template strand has the original 8 repeat units. =nucleotide 核苷酸（nt） 1.Taq and Taq-like enzymes used for PCR have a limited processivity (the number of bases that can be incorporated by each molecule) of 50-60 nt/second 2.Amplicons of 60 nt require more than one enzyme molecule to complete the extension process 3.Every time an enzyme molecule disassociates from the strand, the strand breathes 4.When a new molecule binds to the strands the strands may reanneal out of alignment due to the presence of repeats 5.Enzymes with lower processivitiy yield higher stutter e.g. Stoffel fragment 5-10 nt/second * Peaks that appear multiple repeat units smaller or larger than the true allele Stutter is inversely proportional to the length of the repeat unit Stutter di- tri- tetra- penta- hexanucleotide repeats Each successive stutter product is less intense (allele repeat -1 unit repeat -2 units) Stutter products more than 1 repeat smaller or larger than the allele are generally only seen with dinucleotide repeat loci Longer, uninterrupted stretches of repeats generate higher levels of stutter than shorter stretches Interruptions to the repeat region through mutation impact the level of stutter Stutter percentage is locus specific Stutter percentage is kit specific Stutter peaks can complicate analysis of difficult forensic samples, particularly mixtures * Stutter会影响DNA结果的解释，尤其在DNA样本来自两个或多个个体时。因为stutter产物与真正的等位基因PCR产物大小相同，此时判定一个小峰究竟是含量较少的供者的真实等位基因峰，还是一个相邻等位基因峰的stutter产物是非常困难。 * Possible Causes Too much input DNA Not enough time to add ‘A’ to all products ‘A’ nucleotides may become limiting Incorrect thermal cycling parameters Failur