DNA Quantitation I

WHAT: Shows how much DNA is avail. for ampn.
1. Need to be conc./dil. b4 ampn.
WHY
1. Find optimal amt.
  1. Comm. amp. kits have narrow qty range for optimization
    1. Too little DNA
      1. Allele dropout (missing)
        Heterozyg. assumed as homozyg.
      2. x amp. of some loci (small amp., large not)
        Partial prof. gen.
        Misinterpreted as degraded DNA ➙low level data ➙ x cross threshold ➙ x allele call ➙ x data
      3. Locus imbalance
        Unbalanced amp. of two alleles @ a locus
        Stochastic flux in ratio of two diff alleles
    2. Too much DNA
      1. Stutter
        Offscale data (high rfu); > detection lim.
        10% of rfu, 4bp
      2. Pull-up
        Peak of one color ovrlap
        Same bp; diff col.
      3. Split peak (-A)
        add 3' base tail (poly A tail); so always expect +A; no time to add A, so -A
        Shoulder; Two sep. peaks; split
2. X waste time/res. to repeat process
  1. Some samples wholly consumed, can't be rep.
3. Troubleshooting
  1. Reduce time taken
4. Det. suitable ampn. methods
  1. Target specific types of DNA
    1. Human specific assays
      1. Crime scene x pristine, non-human DNA
      2. DAB std. 9.3 - det. qty. of hDNA
    2. Both autosomal + Y assay (BENEFITS)
      1. In crimes with XY + XX, DNAxx >> DNAxy ➙ low/no amp. of DNAxy ➙ FN
      2. 1 person's DNA >> 2P ➙ single source profile, or 2nd profile too low to use
      3. Y-specific assays target DNAxy rgdless of DNAxx
        Semen present, little/no sperm cells in vag swabs
        Mixture; XX bled more @ scene
        Saliva transfer frm XY ➙ XX (swabbing skin of XX)
        Y-Quantifiler as screening tool: high no. of assault cases; screening proc. lengthy (presumptive + confirmatory); male DNA present ➙ put through screening process

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DNA Quantitation I

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