Long and Accurate Polymerase Chain Reaction (LA PCR).

Abstract

The standard polymerase chain reaction (PCR) is easily capable of amplifying segments of DNA smaller than ∼3 kb in length-sufficient for most purposes, but not enough to amplify an entire mammalian gene, nor even a cDNA of average dimensions. Instead of full-length products, standard PCR amplification of longer templates generates variously sized truncated molecules that appear as unattractive smears on a gel. Long and accurate PCR (LA PCR) addresses the issue in part by using a mixture of two different thermostable DNA polymerases to catalyze the amplification reaction. The first polymerase is an efficient but error-prone workhorse (e.g., Taq), whereas the second, used in much smaller amounts, provides a proofreading 3' → 5' exonuclease function that resects mismatched 3' ends. These improvements generate high yields and accurate copies of long targets.