Recombination and amplification of multiple protions of genomic DNA by a modified polymerase chain reaction

Abstract

A novel method, a modified polymerase chain reaction (mPCR), is reported for in vitro recombination and amplification of two or more regions of DNA located in distinct parts of a genome such as exons of a gene. The reaction consists of three stages. In the first stage, the portions are amplified individually with the aid of four or more primers. Of the primers, two (outside primers) are complementary to 3′-terminal parts of the objective, recombinant DNA, and the others (inside primers) are complementary to 3′-terminal parts of the other portions. Some of the inside primers have extra nucleotide sequences at their 5′-termini which are complementary to 5′-terminal parts of the portions to be recombined. In the second stage, the strands with complementary sequences in their 3′-terminal parts are annealed sequentially to one another and are converted to recombinant DNA with DNA polymerase. In the final stage, the objective DNA is amplified with the excess outside primers. The application of this method for in vitro recombination and amplification of three exons of the gene for human muscle-specific phosphoglycerate mutase is described.