STR interpretation- simple profiles:

Question 1

what is STR profile analysis?

Answer 1

STR profile analysis identifies a person or cell lines unique DNA signature by counting short, repeated DNA sequences called short tandem repeats (STRs) at specific locations, loci, in the genome.

Question 2

simple profile interpretation:

Answer 2

the application of analysis thresholds uses statistics.
objective vs subjective decision making.
cognitive bias.

Question 3

simple profile interpretation steps 1-5:

Answer 3

1. check allele size standard.
2. check positive and negative controls.
3. check allele signal/noise thresholds.
4. check profile and allele balance.
5. check allele stutter.

Question 4

analytical thresholds:

Answer 4

as less DNA is put into a reaction the signal is reduced.
as more DNA is put into a reaction the signal is saturated.
decision needed as to when the profile falls outside the bounds of routine interpretation.

Question 5

threshold:

Answer 5

the threshold is set based on the average noise level across the baseline.
the LOD (limit of detection) is set at 3 standard deviations.
99% of noise should be underneath this.
calculate std deviation for each channel, times by 3 to get 3SD, add on average.

Question 6

allele sizing:

Answer 6

forensic DNA requires accurate sizing of alleles.
the PCR and size separation may be performed at different times using different kits.
quality assurance practices are employed that enable the analyst to minimise inaccuracy.

Question 7

3 ways to achieve accurate sizing of alleles:

Answer 7

size standard run in each well during size separation.
allelic ladders run alongside samples.
DNA controls (positive and negative)

Question 8

size separation:

Answer 8

there are a number of size standards.
each perform same function.
sizing occurs based on standard curve.
relationship between DNA size and migration point.
predict the unknown alleles.

Question 9

bins:

Answer 9

the size standard allows automatic calling as allele bins can be created.
setting an allele bin requires some data on the mean and standard deviation of each allele.

Question 10

allelic ladder.

Answer 10

bin regions are achieved using an allelic ladder. bin windows set to catch allele.
this contains all common alleles.
run at the same time as the unknown or reference sample.

Question 11

data associated with STR profile:

Answer 11

fragment length (bp).
allele repeat size.
RFU of peak height.
RFU of peak area (true/false).

Question 12

profile balance:

Answer 12

DNA profile should be balanced between loci.
homozygotes tend to be higher than heterozygotes.
poor balance can be due to decay or inhibition.

Question 13

heterozygous balance:

Answer 13

an individual will be homozygote or heterozygote at each locus.
if there are 2 copies of an allele in a cell the amplification efficiency during PCR should be the same.
this should mean that the peak heights of both alleles is a heterozygote should be the same (or very similar).

Question 14

a number of effects can occur that can complicate interpretation:

Answer 14

stutter.
mixture.
null allele.
drop out/drop in
causes are different but manifest in similar ways.

Question 15

heterozygote balance threshold set:

Answer 15

threshold set.
area of second allele/ area of first allele
perfect.
perfect balance would be 1.
threshold set from taking multiple measurements.
thresholds set by running internal validation studies.
specific to laboratory.
calculate balance for positive control.

Question 16

mixture:

Answer 16

mixture is a possible explanation. would see multiple alleles present at other loci.
what if the profile does not have more than 2 alleles at other loci.
-primer binding mutation (see null alleles)
-stutter
-random distribution of target material (see drop out).

Question 17

stutter:

Answer 17

stutter products are often observed in the DNA profile. occur at same sizes as allele.
stutter products are formed due to strand slippage during PCR. deletion caused by slippage on the copied (bottom) strand. insertion caused by slippage of the copying (top) strand.
all the human STRs used in forensic analyses are tetra nucleotide. worst is di nucleotide.
the stutter product can contribute to the overall fluorescence of an allele if the alleles are neighbouring.