4) Your laboratory has an Analytical Threshold to separate signal from noise set

Question

4) Your laboratory has an Analytical Threshold to separate signal from noise set at 250RFU and a stutter ratio threshold of 15%. Calculate the stutter ratios for each allele and provide an explanation for why stutter data may not be available for all alleles in this dataset. Peak Area 823 931 631 1217 900 1010 Locus Allele Peak Heterozygote Balance 88% Stutter Height 98 Stutter Ratio Height 300 350 251 480 400 450 D161798 10 THO1 51% 6 9.3 109 100 120 vWA 89% 26 5) Using the allele frequency table and formulas provided at the back of this exam sheet, calculate the genotype frequencies for a genetic profile containing D2 (16-20) with a correction factor, of 0.01. Your qPCR quantification results show that you have recovered a total of 30ng in 200 of AE buffer. First, calculate the concentration of your DNA solution. Second, calculate the volume of DNA to add to the PCR reaction if the total amount required is 2ng. 6)

Explanation / Answer

4) Stutter Ratio = Oa-1 /Oa

Oa-1 is the stutter height,

Oa the parent peak height.

Stutter peaks are the peaks occuring immediately before or after the real peak. This may occur due to polymerase slippage during PCR causing the DNA to be longer or smaller than the actual parent strand. stutter ratios cannot be calculated for sample without stutter peaks.

For D161798 allele 10, stutter ratio:

=stutter peak/peak height

=98/300=0.32

Tho1, 9.3, stutter ratio :

=stutter peak/peak height

= 109/480 =0.227

For vWA,

22 allele, stutter ratio:

= 100/400=1/4=0.25

26 allele, stutter ratio:

=120/450=0.26

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