■ Precision studies. The calculated base pair sizes for STR allele amplification products are measured. All measured alleles should fall within a ± 0.5 bp window around the measured size for the corresponding allele in the allelic ladder.
■ Stutter studies. The percentage of observed stutter at each STR locus is examined by calculating the ratio of the stutter peak area and/or peak height compared to the corresponding allele peak area and height. Stutter values are derived from homozygotes and heterozygotes with alleles separated by at least two repeat units. The upper levels of stutter observed for each locus are then used to develop interpretation guidelines. Because the levels of stutter for each of the 13 CODIS STR loci have been described and usually fall below 10% of the allele peak area and height, many labs just use a standard 15% cut-off for interpreting stutter products. If the stutter peak is below 15% of the allele peak, it is ignored as a biological artifact of the sample. However, if it is above 15% then a possible mixture could be present in the sample (see Chapter 7).
■ Heterozygous peak height balance. The peak heights of the smaller and the larger allele are compared typically by dividing the smaller sized allele peak height by the larger sized allele peak height. In other words, the height of the lower peak in relative fluorescence units (RFU) is divided by the height of the higher peak (in RFU). This peak height ratio is expressed as a percentage. The average heterozygote peak height ratio is usually greater than 90% meaning that a heterozygous individual generally possesses well-balanced peaks. Ratios below 70% are rare in normal, unmixed samples (although primer point mutations can cause one of the alleles to not amplify as well, i.e., a partial null allele - see discussion on null alleles in Chapter 6).
■ Annealing temperature studies. These studies are conducted by running the amplification protocol with the annealing temperature either two degrees above or two degrees below the optimal temperature. Annealing temperature studies are important because thermal cyclers might not always be calibrated accurately and can drift over time if not maintained properly. Thus, an operator might think that the annealing temperature during each cycle is 59°C when in fact the thermal cycler is running hotter at 61°C. If any primers in the multiplex mix are not capable of withstanding slight temperature variation (i.e., they do not hybridize as well), then a locus could dropout or non-specific amplification products could arise.
■ Cycle number studies. The optimal PCR conditions (i.e., denaturing, annealing, and extension temperatures and times) are examined with a reduced number of cycles as well as a higher number of cycles than the standard protocol calls for to evaluate the performance of the STR multiplex system. The sensitivity of detection of alleles for each locus is dependent of course on the quality of input DNA template. The cycle number studies permit a laboratory to determine the tolerance levels of a STR multiplex system with various amounts of DNA template. While a higher number of PCR cycles (e.g., 34 instead of 28) might be able to better amplify very low levels of genomic DNA, the likelihood of nonspecific amplification products arising increases with higher numbers of PCR cycles.
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