Reduction of stutter ratios in short tandem repeat loci typing of low copy number DNA samples

Increased height of stutter peaks is a phenomenon with low copy number (LCN) short tandem repeat (STR) typing that can impact interpretation. An alternative strategy of lowering the annealing/extension temperature (LT) at 56 C was designed to attempt to decrease the heights of stutter peaks. STR typing results were generated in terms of stutter ratios using LT-PCR conditions and compared with data obtained using standard (STD) PCR conditions. DNA samples ranging from 100 to 25 pg were amplified using reagents contained in the AmpFℓSTR^® Identifiler^® PCR Amplification or AmpFℓSTR^® Identifiler^® Plus PCR Amplification kits with 32 or 34 PCR cycles. Stutter ratios decreased by an average of 14.7%, 14.9% and 18.1% at 100, 50 and 25 pg of template DNA under LT conditions compared with those of STD conditions in the Identifiler^® Kit amplified samples. The LT conditions also decreased average stutter ratios by 13.3% compared with those of STD conditions in the Identifiler^® Plus Kit amplified samples. The overall PCR efficiency obtained with STD and LT conditions with the two STR kits was comparable in terms of the number of detected alleles, peak heights and peak height ratios. These results support the hypothesis that a lower temperature annealing/extension step reduces the likelihood of slippage during PCR by enhancing the stability of the DNA polymerase/template DNA complex or the stability of the generated duplex than the conditions of the standard extension step. This stability in turn would result in lower stutter ratios.