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FRNSC421W - QUIZ 4-Questions with Correct Answers/ Verified/ 100% Pass
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FRNSC421W - QUIZ 4-Questions with Correct Answers/ Verified/ 100% Pass
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FRNSC421W - QUIZ 4-Questions with Correct Answers/ Verified/ 100% PassSTR 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
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
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
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)
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
, 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
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
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
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
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
- laser has an excitation wavelength of 505nm, allows for lower noise signals; further away from
excitation wavelength of dyes
- AT calculated by taking 10 SDs from mean of average baseline noise
stochastic threshold (ST) - ✔️✔️minimum peak height (intensity) necessary to eliminate the
possibility of allelic dropout of the second allele of a heterozygote profile
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