Diagnostic and amplification methods using primers having thymine at 3' end to overcome primer-target mismatch at the 3' end

Claim 1

A method for amplifying at least one nucleic acid, said method comprising: A. contacting a specimen comprising a nucleic acid with a primer composition, said primer composition consisting essentially of a primer which is complementary to a sequence of said nucleic acid in every position of said sequence except a single position at the 3' end of said primer, resulting in a single mismatch at said single position between said primer and said nucleic acid sequence, said primer having a nucleotide with a thymine base in the position of said mismatch, and B. substantially simultaneously, contacting said specimen with a DNA polymerase under conditions such that said nucleic acid is amplified in the resulting mixture by forming primer extension products, and denaturation of said primer extension products, said product formation and denaturation being carried out at least five times.

Claim 2

A method for amplifying at least one nucleic acid, said method comprising: A. contacting a specimen comprising a nucleic acid with a primer composition, said primer composition consisting essentially of a primer which is complementary to a sequence of said nucleic acid in every position of said sequence except a single position at the 3' end of said primer, resulting in a single mismatch at said single position between said primer and said nucleic acid sequence, said primer having a nucleotide with a thymine base in the position of said mismatch, and B. substantially simultaneously, contacting said specimen with a DNA polymerase under conditions such that said nucleic acid is amplified in the resulting mixture by forming primer extension products, and denaturation of said primer extension products, said product formation and denaturation being carried out at least five times.

Claim 5

A method for the detection of a target nucleic acid, said method comprising: A. contacting a specimen comprising a nucleic acid with a primer composition, said primer composition consisting essentially of a primer which is complementary to a sequence of said nucleic acid in every position of said sequence except a single position at the 3' end of said primer, resulting in a single mismatch at the single position between said primer and said nucleic acid sequence, said primer having a nucleotide with a thymine base in the position of said mismatch, so as to form hybridized products of said primer and said nucleic acid, B. forming primer extension products in said hybridized products, priming, extending and amplifying said primer extension products using a DNA polymerase in a polymerase chain reaction, C. separating the resulting primer extension products and contacting them with a detection or capture oligonucleotide probe to form a complementary product, and D. detecting the presence of said complementary product as an indication of the presence of said target nucleic acid in said specimen.

Claim 6

A method for the detection of a target nucleic acid, said method comprising: A. contacting a specimen comprising a nucleic acid with a primer composition, said primer composition consisting essentially of a primer which is complementary to a sequence of said nucleic acid in every position of said sequence except a single position at the 3' end of said primer, resulting in a single mismatch at the single position between said primer and said nucleic acid sequence, said primer having a nucleotide with a thymine base in the position of said mismatch, so as to form hybridized products of said primer and said nucleic acid, B. forming primer extension products in said hybridized products, priming, extending and amplifying said primer extension products using a DNA polymerase in a polymerase chain reaction, C. separating the resulting primer extension products and contacting them with a detection or capture oligonucleotide probe to form a complementary product, and D. detecting the presence of said complementary product as an indication of the presence of said target nucleic acid in said specimen.

Claim 13

A method for the detection of at least one predetermined nucleic acid having two complementary strands comprises: A. denaturing the complementary strands of a double-stranded nucleic acid in a biological specimen, B. contacting said specimen with a primer composition, said primer composition consisting essentially of a primer which is complementary to a sequence of one of said nucleic acid strands in every position of said sequence except a single position at the 3' end of said primer, resulting in a single mismatch at said single position between said primer and said nucleic acid strand, said primer having a nucleotide with a thymine base in the position of said mismatch, said specimen also simultaneously contacted with one or more additional primers which are substantially complementary to the other strand of said nucleic acid, so as to form hybridized products of said primers and said nucleic acid strands, C. in the presence of a thermostable polymerase, and the deoxyribonucleotide triphosphates dATP, dCTP, dGTP and dTTP, forming extension products of said primers in said hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of said extension products of said primers, D. separating said primer extension products from the templates on which they were synthesized, E. contacting said separated extension products and said predetermined nucleic acid with additional primers complementary to the strands, resulting in amplification of said nucleic acid to form complementary products, F. separating said primer extension products from the complementary products formed in step E, G. contacting at least one primer extension product separated in step F with an oligonucleotide probe which is labeled for detection or capture and is complementary thereto to form a complementary product of the probe and said primer extension product, and H. detecting the complementary product formed in step G as an indication of the presence of said predetermined nucleic acid in the specimen.

Claim 14

A method for the detection of at least one predetermined nucleic acid having two complementary strands comprises: A. denaturing the complementary strands of a double-stranded nucleic acid in a biological specimen, B. contacting said specimen with a primer composition, said primer composition consisting essentially of a primer which is complementary to a sequence of one of said nucleic acid strands in every position of said sequence except a single position at the 3' end of said primer, resulting in a single mismatch at said single position between said primer and said nucleic acid strand, said primer having a nucleotide with a thymine base in the position of said mismatch, said specimen also simultaneously contacted with one or more additional primers which are substantially complementary to the other strand of said nucleic acid, so as to form hybridized products of said primers and said nucleic acid strands, C. in the presence of a thermostable polymerase, and the deoxyribonucleotide triphosphates dATP, dCTP, dGTP and dTTP, forming extension products of said primers in said hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of said extension products of said primers, D. separating said primer extension products from the templates on which they were synthesized, E. contacting said separated extension products and said predetermined nucleic acid with additional primers complementary to the strands, resulting in amplification of said nucleic acid to form complementary products, F. separating said primer extension products from the complementary products formed in step E, G. contacting at least one primer extension product separated in step F with an oligonucleotide probe which is labeled for detection or capture and is complementary thereto to form a complementary product of the probe and said primer extension product, and H. detecting the complementary product formed in step G as an indication of the presence of said predetermined nucleic acid in the specimen.