US 6,294,323 B1 · Self initiating single primer amplification of nucleic acids

Abstract

A method is disclosed for producing at least one copy of a pair of complementary single stranded polynucleotides. The method comprises forming, in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along each of the complementary single stranded polynucleotides, an extension of a polynucleotide primer. The polynucleotide primer is comprised of at least a sequence of 16 nucleotides terminating at its 3′ end in a 2 to 9 nucleotide sequence (S1), which is complementary with the 3′ ends of both of the complementary single stranded polynucleotides. The polynucleotide primer has at least an 8 nucleotide sequence (S2) that is 5′ of S1, where S2 is 50 to 80% complementary to the nucleotide sequences contiguous with the 3′ ends of the complementary single stranded polynucleotides. The extended polynucleotide primer and the single stranded polynucleotides are then dissociated.

Claims48

Dependent claims

A method of producing at least one copy of a pair of complementary single stranded polynucleotide sequences, said method comprising:

(a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along each of said complementary single stranded polynucleotide sequences, an extension of a single polynucleotide primer, said polynucleotide primer being comprised of at least a sequence of 16 nucleotides terminating at its 3′ end in a 4 to 8 nucleotide sequence (S1) that is complementary with the 3′ ends of both of said complementary single stranded polynucleotide sequences, wherein said polynucleotide primer has a specifically designed sequence of at least 8 nucleotides (S2) that is 5′ of said S1, said S2 being 50 to 80% complementary to each of the nucleotide sequences contiguous with the 3′ ends of said complementary single stranded polynucleotide sequences, and
(b) dissociating said extended polynucleotide primer and said single stranded polynucleotides thereby producing said copy of said single stranded polynucleotide sequences.

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

(a) providing in combination (1) a pair of complementary single stranded polynucleotides having said polynucleotide sequence and its complement, (2) a single polynucleotide primer being comprised of a sequence of 24 to 90 nucleotides terminating at its 3′ end in a 2 to 9 nucleotide sequence (S1) that is complementary with the 3′ ends of both of said polynucleotide sequences and its complement, wherein said polynucleotide primer has a specifically designed sequence of at least 8 nucleotides (S2) that is 5′ of said S1, said S2 being 50 to 80% complementary to each of the nucleotide sequences contiguous with the 3′ ends of said polynucleotide sequence and its complement, (3) nucleoside triphosphates, (4) template dependent polynucleotide polymerase and
(b) incubating said combination under conditions for either wholly or partially sequentially or concomitantly (1) dissociating said polynucleotide sequence and its complement, (2) hybridizing said polynucleotide primer with the sequences at the 3′ end of said polynucleotide sequence and its complement, (3) extending said polynucleotide primer along said polynucleotide sequence and its complement to provide a first complementary pair of extended polynucleotide primers, (4) dissociating said first complementary pair of extended polynucleotide primers from said polynucleotide sequence and its complement, (5) hybridizing single stranded extended polynucleotide primers from said first complementary pair with said polynucleotide primer, (6) extending said polynucleotide primer along said single stranded extended polynucleotide primers to provide a second complementary pair of extended polynucleotide primers, (7) dissociating said second complementary pair of extended polynucleotide primers from said first complementary pair of extended polynucleotide primers, and (8) repeating steps (5)-(7) above thereby producing multiple copies of said polynucleotide sequences.

A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, said analyte having complementary single stranded polynucleotide sequences and nucleotide sequences contiguous with the 3′ ends of said complementary single stranded polynucleotide sequences, said method comprising the steps of:

(a) providing in combination (1) said sample, (2) a polynucleotide primer comprised of at least a sequence of 16 nucleotides terminating at its 3′ end in a 4 to 8 nucleotide sequence (S1) that is complementary with the 3′ ends of both of said complementary single stranded polynucleotide sequences, wherein said polynucleotide primer has a specifically designed sequence of at least 8 nucleotides (S2) that is 5′ of said S1, said S2 being 50 to 80% complementary to each of the nucleotide sequences contiguous with the 3′ ends of said complementary single stranded polynucleotide sequences, (3) nucleoside triphosphates and (4) template dependent polynucleotide polymerase;
(b) incubating said combination under conditions for either wholly or partially sequentially or concomitantly (1) dissociating complementary sequences of said analyte into single stranded polynucleotides, (2) hybridizing said polynucleotide primer with the 3′ end of said single stranded polynucleotide sequences, (3) extending said polynucleotide primer along said single stranded polynucleotide sequences to provide a first complementary pair of extended polynucleotide primers, (4) dissociating said first complementary pair of extended polynucleotide primers from said single stranded polynucleotide sequences, (5) hybridizing single stranded extended polynucleotide primers from said first complementary pair with said polynucleotide primer, (6) extending said polynucleotide primer along said single stranded extended polynucleotide primers to provide a second complementary pair of extended polynucleotide primers, (7) dissociating said second complementary pair of extended polynucleotide primers from said single stranded extended polynucleotide primers, and (8) repeating steps (5)-(7) above,
wherein steps (a) and (b) are performed wholly or partially sequentially or concomitantly;
and
(c) detecting extended polynucleotide primers, the presence thereof indicating the presence of said polynucleotide analyte.

2 more elements

Dependencies are inferred from the claim text.

Self initiating single primer amplification of nucleic acids

Life of this patent

Self initiating single primer amplification of nucleic acids

Life of this patent

Abstract

Claims48