Differences of PCR efficiency between two-step PCR and standard three-step PCR protocols in short tandem repeat amplification

Abstract

The effects on STR typing results, using a two-step PCR protocol that combines the annealing and extension steps, were determined using samples at various DNA concentrations. DNA samples ranging from 62.5 to 500 pg were amplified using the reagents contained within the AmpFlSTR Identifiler PCR Amplification Kit. At 250–500 pg of DNA template, the rates of detecting alleles were close to those found when using three-step PCR (the standard Identifiler protocol) and two-step PCR protocols. The two-step PCR increased by 8.1% and 64.2% the average number of amplified alleles compared with the three-step PCR at 125 pg and 62.5 pg of template DNA, respectively. At 62.5–500 pg of DNA, the average peak heights were 23.3–65.0% higher than heights using the three-step PCR protocol. When two different PCR protocols were applied to old bones, the average number of amplified loci was similar. These results suggested that the two-step PCR protocol can generate more STR alleles in low template DNA samples; however, the method may be limited with samples of very low-quality, i.e., degraded DNA, compared with the three-step protocol. A better understanding of the effects of the two-step PCR protocol on amplification conditions might help researchers improve STR typing.

Keywords:

• DNA typing
• PCR protocol
• short tandem repeat