SELF-FOLDING AMPLIFICATION OF TARGET NUCLEIC ACID
First Claim
1. A method for selective amplification of a target polynucleotide sequence (T), comprising the steps of:
- (a) hybridizing the target polynucleotide sequence (+) with a first composite primer (−
), said first composite primer comprising a 5′
promoter portion (P) and a 3′
target-recognition portion which is complementary to the 3′
end portion of the target polynucleotide sequence;
(b) identifying the 5′
end portion of the target polynucleotide sequence;
(c) extending the 3′
end of the first composite primer and generating a first single-stranded DNA template (−
) comprising the promoter portion (P) and the complementary sequence of the target polynucleotide sequence (Tc), said first single-stranded DNA template comprising a first pair of self-folding segments that are complementary to each other, wherein the promoter portion (P) is at the 5′
end of the first single-stranded DNA template and one of the self-folding segments is at the 3′
end of the first single-stranded DNA template;
(d) allowing the first single-stranded DNA template to self-fold and form a first handle-stem-loop structure, which comprises a 5′
single-stranded handle comprising the promoter portion (P) and a double-stranded stem comprising the first pair of self-folding segments hybridized to each other;
(e) extending the 3′
end of the first handle-stem-loop structure to generate a double-stranded promoter comprising the promoter portion (P) and its complementary sequence (Pc) hybridized to each other; and
(f) initiating transcription from the double-stranded promoter to generate multiple copies of a single-stranded RNA product (+) comprising the target polynucleotide sequence (T).
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Accused Products
Abstract
The application relates generally to methods useful for the selective amplification of one or more target nucleic acid or fragments thereof, as well as compositions and kits comprising said amplification reaction mixtures. More specifically, the application relates to a composite primer that comprises a 5′ promoter portion and a 3′ target-recognition portion which is complementary to the 3′ end portion of a target polynucleotide sequence; and optionally, a means for identifying the 5′ end portion of the target polynucleotide sequence. The amplification reaction mixture comprises at least one handle-stem-loop structure which comprises a 5′ single-stranded handle comprising the promoter portion and a double-stranded stem comprising at least one pair of self-folding segments hybridized to each other, and optionally, a single-stranded loop comprising the sequence between the pair of self-folding segments.
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Citations
20 Claims
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1. A method for selective amplification of a target polynucleotide sequence (T), comprising the steps of:
-
(a) hybridizing the target polynucleotide sequence (+) with a first composite primer (−
), said first composite primer comprising a 5′
promoter portion (P) and a 3′
target-recognition portion which is complementary to the 3′
end portion of the target polynucleotide sequence;(b) identifying the 5′
end portion of the target polynucleotide sequence;(c) extending the 3′
end of the first composite primer and generating a first single-stranded DNA template (−
) comprising the promoter portion (P) and the complementary sequence of the target polynucleotide sequence (Tc), said first single-stranded DNA template comprising a first pair of self-folding segments that are complementary to each other, wherein the promoter portion (P) is at the 5′
end of the first single-stranded DNA template and one of the self-folding segments is at the 3′
end of the first single-stranded DNA template;(d) allowing the first single-stranded DNA template to self-fold and form a first handle-stem-loop structure, which comprises a 5′
single-stranded handle comprising the promoter portion (P) and a double-stranded stem comprising the first pair of self-folding segments hybridized to each other;(e) extending the 3′
end of the first handle-stem-loop structure to generate a double-stranded promoter comprising the promoter portion (P) and its complementary sequence (Pc) hybridized to each other; and(f) initiating transcription from the double-stranded promoter to generate multiple copies of a single-stranded RNA product (+) comprising the target polynucleotide sequence (T). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification