Detection of specific sequences in nucleic acids

A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid, which method comprises:

• (a) treating the denatured nucleic acid with a diagnostic probe complementary to a diagnostic portion of the target sequence, and with a contiguous probe complementary to a nucleotide sequence in the target sequence which is contiguous with the diagnostic portion, under conditions of stringency wherein the diagnostic probe and the contiguous probe would become hybridized to the denatured nucleic acid if the target sequence were present in the sample, said conditions of stringency such that the diagnostic probe remains bound substantially only to sample nucleic acid containing the target sequence, the diagnostic probe and the contiguous probe being of sufficient length to be able to stably hybridize to the target sequence long enough so that the diagnostic probe and the contiguous probe can be covalently attached to each other, and wherein at least one of the diagnostic and contiguous probes is labeled;
• (b) covalently attaching the diagnostic probe to the contiguous probe after the sample has been treated with the diagnostic probe and the contiguous probe in step (a) to produce a target probe complementary to the target sequence;
• (c) removing the probes not attached in step (b);
• and
• (d) testing for the presence of label in the sample after step (c).

A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid using a diagnostic probe which is complementary to a diagnostic portion of the target sequence and a contiguous probe which is complementary to a portion of said target sequence which is contiguous with said diagnostic portion, wherein the diagnostic probe and the contiguous probe are of sufficient length that they can be stably hybridized to the target sequence long enough to be covalently attached to each other, and wherein one of the diagnostic probe and the contiguous probe will remain hybridized to the target sequence under a higher stringency than will the other, comprising the steps of:

• (a) hybridizing the one of the diagnostic and contiguous probes which will remain bound to its complementary portion of the target sequence under the higher stringency, said one of the diagnostic and contiguous probes hereinafter called the HS probe;
• (b) after Step (a) hybridizing the other of said diagnostic and contiguous probes which will remain bound to its complementary portion in the target sequence under conditions of lower stringency, said other of said diagnostic and contiguous probes hereinafter called the LS probe;
• (c) removing a substantial portion of said diagnostic probe which is not perfectly bound;
• (d) covalently attaching said LS probe and said HS probe where they are bound adjacent to each other to form a target probe complementary to said target sequence;
• (e) removing probe not attached in step (d);
• and
• (f) testing for the presence of target probe in the sample remaining after Step (e).

A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid, the steps comprising:

• (a) treating the denatured nucleic acid with a diagnostic probe complementary to a diagnostic portion of the target sequence, and with a contiguous probe complementary to a nucleotide sequence in the target sequence which is contiguous with the diagnostic portion, under conditions wherein the diagnostic probe and the contiguous probe would become hybridized to the denatured nucleic acid if the target sequence were present in the sample, wherein at least one of the diagnostic and contiguous probes is labeled, wherein the diagnostic and contiguous probes can be covalently attached, wherein the diagnostic probe and the contiguous probe are of sufficient length to be stably hybridized to the target sequence long enough to be covalently attached to each other while they are hybridized to the target sequence, and wherein conditions of stringency are controlled so that said diagnostic probe is specifically bound;
• (b) after step (a), treating the sample to covalently attach the diagnostic probe to the contiguous probe where they are adjacent on the target sequence, to produce a target probe complementary to the target sequence;
• (c) removing the target probe formed in step (b) from the sample to form a second sample containing the target probe;
• and
• (d) testing for the presence of label in the second sample.