Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof
First Claim
1. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:
- (a) hybridizing a composite primer to a target polynucleotide, wherein the composite primer comprises an RNA portion and a 3′
DNA portion, the 3′
DNA portion comprising a 3′
most nucleotide, such that the 3′
most nucleotide of the 3′
DNA portion hybridizes from 1 to about 10 nucleotides from the sequence of interest;
(b) extending the composite primer with DNA polymerase under conditions that permit primer extension, whereby a primer extension product is produced; and
(c) cleaving the RNA portion of the primer extension product of(b) with an enzyme that cleaves RNA from an RNA/DNA hybrid such that the cleaved primer extension product dissociates from the target polynucleotide, wherein the primer extension product is of a size that when the RNA is cleaved the cleaved primer extension product dissociates from the target polynucleotide under essentially the same conditions as those for primer extension, whereby multiple copies of the sequence of interest are produced.
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Abstract
The present invention provides novel isothermal methods of generating multiple copies of, detecting and/or quantifying nucleic acid sequences of interest based on limited primer extension or attachment of oligonucleotide pairs using composite RNA/DNA primers. Methods for generating multiple copies of and/or detecting and/or quantifying nucleic acid sequences, wherein products of primer extension or attachment of oligonucleotide pairs comprising a cleavable portion are generated, and wherein cleavage of the products results in dissociation of cleaved products from target polynucleotides, are provided. The invention further provides compositions, kits and systems for practicing these methods.
137 Citations
79 Claims
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1. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:
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(a) hybridizing a composite primer to a target polynucleotide, wherein the composite primer comprises an RNA portion and a 3′
DNA portion, the 3′
DNA portion comprising a 3′
most nucleotide, such that the 3′
most nucleotide of the 3′
DNA portion hybridizes from 1 to about 10 nucleotides from the sequence of interest;
(b) extending the composite primer with DNA polymerase under conditions that permit primer extension, whereby a primer extension product is produced; and
(c) cleaving the RNA portion of the primer extension product of(b) with an enzyme that cleaves RNA from an RNA/DNA hybrid such that the cleaved primer extension product dissociates from the target polynucleotide, wherein the primer extension product is of a size that when the RNA is cleaved the cleaved primer extension product dissociates from the target polynucleotide under essentially the same conditions as those for primer extension, whereby multiple copies of the sequence of interest are produced. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of determining whether a nucleic acid sequence of interest is present or absent in a sample, said method comprising the steps of:
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(a) hybridizing a composite primer to a target polynucleotide, wherein the composite primer comprises an RNA portion and a 3′
DNA portion, the 3′
DNA portion comprising a 3′
most nucleotide, such that the 3′
most nucleotide of the DNA portion of the composite primer hybridizes from 1 to about 10 nucleotides from the sequence of interest;
(b) extending the composite primer with DNA polymerase under conditions that permit primer extension, whereby a primer extension product comprising a detectable identifying characteristic is produced if the sequence of interest is present; and
(c) cleaving the RNA portion of the primer extension product of (b), if any, with an enzyme that cleaves RNA from an RNA/DNA hybrid such that the cleaved primer extension product dissociates from the target polynucleotide, wherein the primer extension product is of a size that when the RNA is cleaved the cleaved primer extension product dissociates from the target polynucleotide under essentially the same conditions as those for primer extension, whereby detection of the cleaved primer extension product comprising the detectable identifying characteristic indicates the presence of the sequence of interest. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 73, 74, 75)
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37. A method of generating multiple copies of a nucleic acid sequence of interest comprising:
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incubating a reaction mixture, said reaction mixture comprising;
(a) a target polynucleotide;
(b) a composite primer that hybridizes to the target polynucleotide, said composite primer comprising an RNA portion and a 3′
DNA portion, the 3′
DNA portion comprising a 3′
most nucleotide, such that the 3′
most nucleotide of the 3′
DNA portion of the primer hybridizes from 1 nucleotide to about 10 nucleotides from the sequence of interest;
(c) a DNA polymerase; and
(d) an enzyme that cleaves RNA from an RNA/DNA hybrid, wherein the incubation is under conditions that permit primer hybridization, primer extension and RNA cleavage, such that a primer extension product is produced, and wherein the primer extension product is of a size such that cleavage of RNA from the primer extension product results in dissociation of the cleaved primer extension product from the target polynucleotide. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49)
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50. A method of determining whether a nucleic acid sequence of interest is present or absent in a sample comprising incubating a reaction mixture, said reaction mixture comprising:
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(a) a target polynucleotide;
(b) a composite primer that hybridizes to the target polynucleotide, said composite primer comprising an RNA portion and a 3′
DNA portion, the 3′
DNA portion comprising a 3′
most nucleotide, such that the 3′
most nucleotide of the 3′
DNA portion of the primer hybridizes from 1 nucleotide to about 10 nucleotides from the sequence of interest;
(c) a DNA polymerase; and
(d) an enzyme that cleaves RNA from an RNA/DNA hybrid, wherein the incubation is under conditions that permit primer hybridization, primer extension to generate a primer extension product comprising a detectable identifying characteristic, and RNA cleavage, such that the primer extension product comprising a detectable identifying characteristic is produced, and wherein the primer extension product is of a size such that cleavage of RNA from the primer extension product results in dissociation of the cleaved primer extension product from the target polynucleotide, whereby detection of the cleaved primer extension product comprising the detectable identifying characteristic indicates presence of the nucleotide sequence of interest. - View Dependent Claims (51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72)
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76. A method for identifying an altered sequence of interest in a sample comprising incubating a reaction mixture, said mixture comprising:
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(a) a target polynucleotide;
(b) a composite primer that hybridizes to the target polynucleotide, said composite primer comprising an RNA portion and a 3′
DNA portion, the 3′
DNA portion comprising a 3′
most nucleotide, such that the 3′
most nucleotide of the 3′
DNA portion of the primer hybridizes from 1 nucleotide to about 10 nucleotides from the altered sequence of interest;
(c) a DNA polymerase; and
(d) an enzyme that cleaves RNA from an RNA/DNA hybrid, wherein the incubation is under conditions that permit primer hybridization and primer extension to generate a primer extension product comprising a detectable identifying characteristic, and RNA cleavage, such that a primer extension product comprising a detectable identifying characteristic is produced, and wherein the primer extension product is of a size that when RNA is cleaved from the primer extension product, the cleaved primer extension product dissociates from the target polynucleotide, whereby the cleaved primer extension product is characterized to identify the altered sequence of interest. - View Dependent Claims (77)
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78. A method of identifying an altered sequence of interest in a sample, said method comprising incubating a reaction mixture, said reaction mixture comprising:
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(a) a target polynucleotide;
(b) a composite primer that hybridizes to the target polynucleotide, said composite primer comprising an RNA portion and a 3′
DNA portion, the 3′
DNA portion comprising a 3′
most nucleotide, such that the 3′
most nucleotide of the 3′
DNA portion of the primer hybridizes from 1 nucleotide to about 10 nucleotides from the altered sequence of interest;
(c) a DNA polymerase; and
(d) an enzyme that cleaves RNA from an RNA/DNA hybrid, wherein the incubation is under conditions that permit primer hybridization, and primer extension to generate a primer extension product comprising a detectable identifying characteristic, and RNA cleavage, such that the primer extension product comprising a detectable identifying characteristic is produced, and wherein the primer extension product is of a size that when RNA is cleaved from the primer extension product, the cleaved primer extension product dissociates from the target polynucleotide, and wherein production of detectably fewer cleaved primer extension products from the target as compared to the amount of cleaved primer extension products produced from a reference template comprising the sequence of interest indicates that the target polynucleotide contains an altered sequence of interest. - View Dependent Claims (79)
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Specification