FRNSC421W - QUIZ 4 EXAM
QUESTIONS AND ANSWERS
2024
1. STR fluorescence incorporation: incorporates fluorescent dyes on PCR
primers as labels for detection of target alleles
- one of two primers at each locus is labeled
- only one ssDNA fragment will be detected for each allele
- allows for analysis of overlapping DNA fragments from different loci and ILS
control for lane-to-lane variability
2. Fusion 6C matrix file: runs individual dyes to visualize intensity of fluorescent
peaks
- intensity of peaks allows for an estimate of how much fluorescence bleeds into
other channels
- can quantify and subtract bleed-through from neighboring channels
3. mobility modifying linkers: allow for amplified alleles in a pair of closely spaced
STR loci to be shifted away from one another
- placed between primer and dye to change migration pattern of amplified product
- allows loci to be spaced properly, prevents overlapping size ranges
4. internal lane standard (ILS): run with each sample in each capillary to correct
capillary-to-capillary migration anomalies
- each DNA fragment sized by an ILS, all run in the same capillary with different
dyes- migration anomalies often seen on edge of gel; move faster because of
increased heat, i.e. 'curling bases'
- detection window excites dyes, CCD camera captures emission as a picture
- creates 'standard' for fragments to be sized by scan # (x-axis) vs size in bps (y-
axis), where larger DNA fragment has larger scan # (more pictures taken)
1/7
, 5. local southern method: uses a virtual standard curve using three data points 1)
generate virtual standard curve using 2 ILS fragments above and 1 ILS fragment
below where unknown DNA falls on curve, extrapolate bp value
2) generate virtual standard curve using 1 ILS fragment above and 2 ILS fragments
below where unknown DNA falls on curve; extrapolate bp value
3) average two values
6. multi-color fluorescence genotyping: the ILS run with the Fusion allelic ladder
allows for ladder fragments to be sized and placed into sizing/allele bins
- unknown DNA fragments sized with their own ILS can be placed into bins
and designated an allele number - unshaded bins are partial repeats
- fragments of DNA with all six dyes are run through the same capillary;
software separates fragments by dye color
7. allele designations: based on ISFG rules
- whole integers representing the # of tandem repeats with specific
nomenclature for partial/incomplete motifs
- slightly altered repeats due to migration to different positions during
electrophoresis- ex: a 31.2 allele at D21
8. peak migration: peaks in fluorescence are given a numerical allele value using
the combination of the ILS and the allelic ladder
- characterized by the labeling color of the locus-specific primers and the length of
the amplified fragment
9. off-ladder (OL) alleles: may be due to migration issues
- compare size of allele with nearby alleles in allelic ladder to determine
appropriate numerical value
- for alleles between ladders, assess whether allele is homo- or heterozygous-
'new' OL alleles should be rerun; common OL alleles are listed that do not have to
be rerun
10. analytical threshold (AT): minimum peak height (intensity) necessary to
identify and differentiate potential true alleles from instrument noise
- non-reproduceable noise peaks may be detected above AT
- using a high AT increases risk of allelic loss
2/7
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