Long PCR: selective suppression by restriction endonuclease digestion.

The polymerase chain reaction (PCR) has become the most widely used technique for the manipulation of DNA with applications for cDNA cloning, gene cloning, polymorphism detection, mutagenesis and allele-specific diagnosis (8). All of those applications have been extended significantly by the recent development of “long” PCR, a technique that makes it possible to amplify DNA fragments up to 40 kb in length (2,3). Included among important factors for practical use of the PCR are high specificity and high yield. Many different approaches have been used in an attempt to increase both reaction yield and specificity. For example, considerable effort has been expended on the optimal design of PCR primers and on modifications of reaction conditions, such as the use of a so-called “hot start”, the initial addition of reagents at an elevated temperature (4,5). Nonspecific amplification is a particular problem in PCR when it is not possible to design optimal primers, either as a result of limited knowledge of the DNA sequence to be amplified or in situations in which multiple copies of similar sequences are present in the template DNA. We have recently attempted to deal with such a situation by using an approach that we term “selective suppression” by restriction endonuclease digestion. That approach combines, in a single reaction tube, restriction endonuclease digestion and a hot start for PCR. This technique eliminates template DNA sequences that may result in nonspecific amplification by restriction digestion performed immediately before PCR; so it is unnecessary to design highly specific primers. Furthermore, since template sequences that may compete during PCR have been removed before the reaction, it is also possible to obtain increased yields of the desired amplification product. Reagents required for restriction digestion of template DNA are initially separated from the PCR reactants by a wax barrier. However, after hot start of PCR, final concentrations of all reactants are optimal for performance of PCR. Selective suppression of long PCR will be illustrated by its use to amplify individual members of a group of at least three human chromosome 16 phenol sulfotransferase (PST) genes, STP1, STP2 and STM (1,7). With the exception of regions that contain Alu sequences, these three genes display very high sequence homology, even within introns (1,7). A major problem associated with the use of PCR to amplify these three genes with genomic DNA as a template has been the nonspecific amplification of sequence fragments from more than one gene. The use of selective suppression makes possible higher yields of specific amplification products by initial restriction digestion that leaves only one of the PST gene sequences intact, which is followed by performance of long PCR in the same reaction tube. When long PCR (GeneAmp® XL PCR Kit; PerkinElmer, Norwalk, CT, USA) is performed with human genomic DNA as a template, segments from all three PST genes are amplified simultaneously by primers F2 (5′-AATGCCCGCAACAGTGCCTGCTGCATAGAG-3′) and R2 (5′-CTCCGCATAGTCCGCATCG-