Method for sequencing both strands of a double stranded dna in a single sequencing reaction

Abstract

A method is presented which uses a unique opposite strand joining strategy during PCR of an original DNA to generate a product which, when sequenced with a single sequencing primer yields the sequence of both strands of the original DNA. The PCR primers include 1) a modified oligomer corresponding to the 5′ end of a first strand of the DNA to be amplified wherein said modified oligomer includes the reverse complementary sequence to a sequence within said first strand of DNA and a specific PCR priming sequence which will specifically hybridize to a portion of the DNA to be amplified and 2) a second oligomer corresponding to the 5′ end of the second strand of the DNA to be amplified and which contains the priming sequence for the second strand of the DNA and will specifically hybridize to a portion of the DNA to be amplified. During PCR an intermediate product is formed where one end of one strand loops around to hybridize to its complement on the same strand. This results in a hairpin structure which elongates using its own strand as a template to form a double sized product that contains the sequence of both original strands. Upon denaturation this yields single strands with the single strands having the sequence of both of the original strands included in tandem. Sequencing these single strands using a single primer, e.g., a primer complementary to the second oligomer, yields the sequences of both strands of the DNA of interest.