Endonuclease-mediated long PCR and its application to restriction mapping.

Abstract

The polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of "long" PCR, a technique that makes it possible to amplify DNA fragments up to 40 kb in length. This article describes two novel applications of the long PCR technique, one which simplifies restriction mapping and another which enhances amplification specificity and yield. The same primers used to perform the long PCR amplification can be used as probes to perform restriction mapping of the DNA fragment amplified. Restriction digestion performed prior to long PCR amplification can be used to selectively suppress the amplification of members of families of closely related DNA sequences, thereby making it possible to selectively amplify one of a group of highly homologous sequences. These two complimentary techniques, both involving use of the long PCR paired with restriction digestion, have potential application in any laboratory in which PCR is performed.