STR Analysis Flashcards
for heterozygous loci (equation)
P = 2pq
P
probability
p & q
frequencies of alleles in a given population
find the probability: for the locus D3S1358 & individual is 15,18 with frequencies of 0.2825 & 0.1450 respectively
P = 2(0.2825)(0.1450) = 0.0819
3 steps in data interpretation
- asses the internal size standards (ISS), allelic ladders & controls
- assess each sample for the presence of extraneous peaks & determine if they may interfere with the interpretation process
- assess the data from each sample
Internal Size Standards (ISS)
- contains DNA fragments of known sizes that provide reference points determining the length of a samples DNA fragments
- in general, the peaks or bands from as ISS are uniformity or miscalled peaks cans indicate problems with the sample, injection &/or run
allelic ladder
contain the more common alleles in general population for specific chromosomal locations
positive control
- positive controls are included in most commercial DNA analysis kits
- amplified with each batch of samples
- show the expected alleles
- if no peaks are seen, this may indicate problem with amplification or injection
ISS negative control
see nothing
ISS data evaluation
peak height percentage
Peak Height Percentage
- heterozygous peaks within a locus should be similar in height to each other
- heterozygous alleles have peak heights that are within 70% of each other
peak height percentage: degraded DNA
may have peak height percentages much lower than 70%
how to calculate Peak Height Percentage
(small/big) * 100
sex identification with Amelogenin
6 bp deletion on the X chromosome
the following may indicate a mixture:
- the presence of more than 2 alleles at any one locus
- imbalanced &/or unexpected peak height percentage
what to do after indication its a mixture
it may be helpful to use the case info &/or sample type to aid in the interpretation
minor/major contributors
determining genotype of major contributor is easier than determining the minor contributor
with known contributors
- subtract the contribution of the known donor from the mixed profile
- common aproach for intimate samples
unknown contributors
- 2 or more people
- commonly use the approach of estimating percent contribution or percent peak height ratio
calculating percent contribution
- assuming there are 2 donors, the mixture proportions are estimated by summing the peak heights of the 2 major or minor alleles & dividing by the peak heights of all four alleles
- this is multiplied by 100 to provide a percentage contribution
reporting guidelines
- inclusion/match
- exclusion/nonmatch
- inconclusive/uninterpretable
- no results
reporting guidelines: inclusion/match
all loci match between a questioned & known sample
reporting guidelines: exclusion/nonmatch
one locus does not match
reporting guidelines: inconclusive/uninterpretable
a potential determination with complex mixtures or degraded samples
reporting guidelines: no results
no discernable allelic activity observed at a locus
common interpretation problems
- thresholds
- spurious peaks
- stutter
- degradation
- allelic drop out
common interpretation problems: thresholds
- established peak-height levels for scoring alleles
- only if fluorescence exceeds this value
- each laboratory must set its own as part of its validation procedure
common interpretation problems: spurious peaks
- dye blobs, noise, spikes
- not reproducible - run the sample again
common interpretation problems: stutter
- by-product of the amplification of STR loci - a minor product, typically one repeat smaller than the primary allele is generated
- due to slipped strand mispairing during the amplification process
common interpretation problems: degradation
can result in a partial profile or null allele
common interpretation problems: allelic dropout
- the initial input quantity of DNA is too low, resulting in the failure to amplify one or more alleles in the sample
- a mutation in the primer binding site is present, which causes a failure in the amplification of the allele
allelic dropout
a sample is typed & one or more alleles are not present
