Rapid PCR of STR markers: Applications to human identification

Multiplex PCR with fluorescently labeled primers has been an essential method for the amplification of short tandem repeats used in human identify testing. Within the STR workflow of extraction, quantitation, amplification, separation, and detection, multiplex PCR is commonly identified as the bottleneck in the process. The time requirement of up to three hours to complete 28-30 cycles of multiplex PCR for STR genotyping is the greatest amount of time required for a single step within the process. The historical use of commercially available thermal cyclers and heat stable polymerases may have given the impression that large multiplex will always require long PCR cycling times to ensure that all of the varying sized targets (typically 100-400 bp) can be amplified in a balanced manner throughout the multiplex. However, with the advent of improved polymerases and faster thermal cyclers the time required for the amplification of large STR multiplexes is no longer on the order of three hours, but as little as 14 min. Faster amplification times can be performed while retaining the balance and integrity of large multiplex PCRs by implementation of alternate polymerases and thermal cyclers. With the reduction in PCR cycling times there has also been an impact on the development of integrated and microfluidics devices which employ the use of reduced or rapid thermal cycling protocols as part of their integration. Similarly, PCR inhibitor resistant polymerases can also reduce overall STR processing times for reference samples by eliminating the need for DNA extraction and purification that is additionally implemented within the development of integrated DNA typing devices.