Patent ID: 6027913
Filing Date: 2000-02-22
Classification: C12N,C12Q

Abstract:
A method of synthesizing a polypeptide encoded for by a sequence of interest contained in a nucleic acid molecule which comprises:(a) obtaining a sample of the nucleic acid molecule which contains the sequence of interest;(b) if the nucleic acid molecule is a single-stranded RNA molecule, reverse transcribing the sample from step (a) so as to prepare a sample containing a DNA molecule which contains a sequence complementary to the sequence of interest;(c) treating the sample from step (a) if the nucleic acid molecule is a DNA molecule or the sample from step (b) if the nucleic acid molecule is a single-stranded RNA molecule so as to obtain a further sample containing a single-stranded DNA molecule which contains a sequence complementary to the sequence of interest;(d) contacting the further sample from step (c) under hybridizing conditions with one oligonucleotide primer which includes at least (a) a promoter and (b) a nucleic acid sequence present within the nucleic acid molecule which contains the sequence of interest with a translation initiation signal 3' to the promoter, which primer sequence is located adjacent to, and 5' of, the sequence of interest, so that the oliqonucleotide primer hybridizes with the single-stranded DNA molecule which contains the sequence complementary to the sequence of interest;(e) treating the resulting sample containing the single-stranded DNA molecule to which the oligonucleotide primer is hybridized from step (d) with a polymerase under polymerizing conditions so that a DNA extension product of the oligonucleotide primer is synthesized, which DNA extension product contains the sequence of interest;(f) treating the sample from step (e) so as to separate the DNA extension product from the single-stranded DNA molecule on which it was synthesized and thereby obtain single-stranded DNA molecules;(g) contacting the resulting sample from step (f) containing the sinqle-stranded DNA molecule which contains the sequence complementary to the sequence of interest under hybridizing conditions, with the oligonucleotide primer so that the oligonucleotide primer hybridizes with the single-stranded DNA molecule present in the sample which contains the sequence complementary to the sequence of interest;(h) treating the sample containing the single-stranded DNA molecule to which the oligonucleotide primer is hybridized from step (g) with a polymerase so as to synthesize a further DNA extension product containing the sequence of interest;(i) repeating steps (f) through (h), as desired;(j) contacting the sample from step (i) with an RNA polymerase which initiates polymerization from the promoter present, under polymerizing conditions, so as to obtain multiple RNA transcripts of each DNA extension product which contains the sequence complementary to the sequence of interest; and(k) translating the RNA transcripts of step (j) to produce the polypeptide or fragment thereof encoded for by the nucleic acid molecule.