US 5,508,178 A · Nucleic acid amplification using single primer

Abstract

A method is disclosed for determining the presence of a polynucleotide analyte in a sample suspected of containing the analyte. The method comprises (a) forming as a result of the presence of an analyte a single stranded polynucleotide comprising a target polynucleotide binding sequence flanked by first and second polynucleotide sequences that differ from the sequence of the analyte or a sequence complementary to the analyte sequence, (b) forming multiple copies of the single stranded polynucleotide, and (c) detecting the single stranded polynucleotide. Also disclosed is a method of producing at least one copy of a single stranded polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'-end of which has at least a 10 base sequence hybridizable with a second sequence flanking the 3'-end of the single stranded polynucleotide, the second sequence being partially or fully complementary with at least a 10 base first sequence flanking the 5' end of the single stranded polynucleotide, (b) dissociating the extended polynucleotide primer and the single stranded polynucleotide, (c) repeating step a and (d) dissociating the extended polynucleotide primer and the copy of the single stranded polynucleotide.

Claims25

Dependent claims

A method of producing at least one copy of a single stranded polynucleotide which comprises:

(a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along a single stranded polynucleotide, flanked at the 5'-end and the 3'-end, respectively, by a first flanking sequence and a second flanking sequence, an extension of a polynucleotide primer at least the 3'-end of said polynucleotide primer having at least a 10 base sequence hybridizable with a 10 base sequence of said second flanking sequence, said 10 base sequence of said second flanking sequence being at least partially complementary, and thus hybridizable, with said first flanking sequence;
(b) dissociating said extended polynucleotide primer and said single stranded polynucleotide;
and
(c) repeating step a wherein said polynucleotide primer hybridizes to and is extended along (1) said single stranded polynucleotide and (2) said extension of said polynucleotide primer.

1 more element

A method of producing multiple copies of a polynucleotide sequence, which comprises:

(a) providing in combination (1) a single stranded polynucleotide having said polynucleotide sequence and being flanked at each end by at least partially complementary first and second flanking sequences, (2) a polynucleotide primer at least a 10 base portion of which at its 3'-end is hybridizable to a 10 base sequence of that member of said first and second flanking sequences that is at the 3'-end of said single stranded polynucleotide, said 10 base sequence being hybridizable to a 10 base sequence of that member of said first and second flanking sequences that is at the 5'-end of said single stranded polynucleotide, (3) nucleoside triphosphates, and (4) template dependent polynucleotide polymerase and
(b) incubating said combination under conditions for either wholly or partially sequentially or concomitantly (1) dissociating said single stranded polynucleotide from any complementary sequences, (2) hybridizing said polynucleotide primer with the flanking sequence at the 3' end of said single stranded polynucleotide, (3) extending said polynucleotide primer along said single stranded polynucleotide to provide a first extended polynucleotide primer, (4) dissociating said first extended polynucleotide primer and said single stranded polynucleotide, (5) hybridizing said first extended polynucleotide primer with said polynucleotide primer, (6) extending said polynucleotide primer along said first extended polynucleotide primer to provide a second extended polynucleotide primer, (7) dissociating said second extended polynucleotide primer from said first extended polynucleotide primer, and (8) repeating steps (5)-(7) above.

A method of producing multiple copies of a polynucleotide sequence in a polynucleotide, said sequence being flanked at each end by a different member of a pair of flanking sequences that are at least partially complementary to each other, which comprises:

(a) combining said polynucleotide with a single polynucleotide primer having at least a terminal sequence at its 3'end at least partially complementary to and hybridizable with at least a portion of the member of said pair of flanking sequences at the 3'-end of said polynucleotide sequence, said portion being hybridizable with the member of said pair of flanking sequences at the 5'-end of said polynucleotide sequence, nucleoside triphosphates, and template dependent polynucleotide polymerase,
(b) incubating said combination under conditions for either wholly or partially sequentially or concomitantly (1) dissociating said polynucleotide sequence from any sequence with which it is hybridized to provide a single stranded polynucleotide, (2) hybridizing said polynucleotide primer with the flanking sequence at the 3'-end of said single stranded polynuycleotide, (3) extending said polynucleotide primer along said single stranded polynucleotide to provide an extended polynucleotide primer, (4) dissociating said first extended polynucleotide primer and said single stranded polynucleotide, (5) hybridizing said first extended polynucleotide primer with said polynucleotide primer, (6) extending said polynucleotide primer along said first extended polynucleotide primer to provide a second extended polynucleotide primer, (7) dissociating said second extended polynucleotide primer from said first extended polynuycleotide primer, and (8) repeating steps (5)-(7) above.

Dependencies are inferred from the claim text.

Nucleic acid amplification using single primer

Life of this patent

Nucleic acid amplification using single primer

Life of this patent

Abstract

Claims25