Optimization strategies for the polymerase chain reaction

The GeneAmp polymerase chain reaction (PCR) process has now become a key procedure in molecular biology research laboratories. The PCR technique is an in vitro method in which genomic or cloned target sequences are specifically enzymatically amplified as directed by a pair of oligonucleotide primers. This technique has been quite robust in the hands of the majority of researchers and is extremely flexible, as evidenced by the increasing number of related PCR formats (i.e., inverse PCR, anchored PCR, asymmetric PCR, labeled primer PCR and RNA-PCR). Today's applications include direct sequencing, genomic cloning, DNA typing, detection of infectious microorganisms, site-directed mutagenesis, prenatal genetic disease research, and analysis of allelic sequence variations. Scientists at Cetus and Perkin-Elmer have collaborated for several years to better understand the interacting biochemical and biophysical parameters which affect PCR optimization. Following are many of the current recommendations, offered with the caveat that our understanding of the PCR process is continually evolving.