Back to results
US 12,577,615 B2Granted
Methods for the detection of a nucleic acid
AssigneeRHODX, Inc.
InventorCharles Rodi
Life of this patent
Estimating the term…
Abstract
In alternative embodiments, provided are primer-based nucleic acid amplification methods capable of detecting the presence of a nucleic acid sequence including at nucleotide level resolution by means of the production of a reporter. In alternative embodiments, provided are hybridization-based nucleic acid detection methods capable of single nucleotide resolution. In alternative embodiments, provided are methods for detecting a target nucleic acid sequence in a sample, which can comprises a biological sample, and optionally the biological sample comprises a cell free nucleic acid (cfNA), optionally a cell-free DNA (cfDNA) or a cell-free RNA (cfRNA), or a circulating cell-free DNA (ccfDNA) or a circulating cell-free RNA (ccfRNA), or an environmental DNA (eDNA) or an environmental RNA (eRNA).
Claims29
A method for detecting a target nucleic acid sequence, comprising:
- (a) providing or having provided a sample,
- (b) providing or having provided a first synthetic nucleic acid polynucleotide primer,
- wherein the first synthetic nucleic acid polynucleotide primer comprises:
- (i) nucleotide residues for base pairing pair or selectively binding or annealing to the target nucleic acid sequence under physiologic conditions, and
- (ii) one or more 5′ nucleotide residues that are not complementary to the target nucleic acid;
- (c) providing or having provided a DNA polymerase, and an exonuclease or endonuclease capable of recognizing the one or more 5′ nucleotide residues of the first synthetic nucleic acid polynucleotide primer are not complementary to the target nucleic acid when incorporated in a double-stranded oligonucleotide, and based on this recognition the exonuclease or endonuclease cleaves the double-stranded oligonucleotide;
- (d) contacting the first synthetic nucleic acid polynucleotide to the sample under conditions wherein the first synthetic nucleic acid polynucleotide is capable of annealing to the target nucleic acid;
- (e) extending the length of the first synthetic nucleic acid polynucleotide primer in a 5′ to 3′ direction using the DNA polymerase using the target nucleic acid as a template, thereby generating an extended first synthetic nucleic acid polynucleotide primer;
- (f) providing or having provided a reverse synthetic nucleic acid primer capable of binding or selectively annealing to the 3′ end of the extended first synthetic nucleic acid polynucleotide primer, and binding or selectively annealing the reverse synthetic nucleic acid to the extended first synthetic nucleic acid polynucleotide primer;
- (g) extending the length of the reverse synthetic nucleic acid in a 5′ to 3′ direction using the DNA polymerase using the extended synthetic nucleic acid polynucleotide primer as a template, and incorporating in the extended length the one or more 5′ nucleotide residues that was not complementary to the target nucleic acid, thereby generating a double-stranded oligonucleotide comprising the one or more 5′ nucleotide residues that was not complementary to the target nucleic acid;
- and
- (h) contacting the double-stranded oligonucleotide with the exonuclease or endonuclease, thereby cleaving or separating the one or more 5′ nucleotide residues that was not complementary to the target nucleic acid from the double-stranded oligonucleotide, thereby generating one or more fragments of single-stranded nucleotide,
- wherein the generation or presence the one or more fragments of single stranded nucleotide indicates the presence of the target nucleic acid.
10 more elements
A method for detecting a target nucleic acid sequence, comprising:
- (a) providing or having provided a sample comprising a target nucleic acid;
- (b) providing or having provided a synthetic nucleic acid polynucleotide primer,
- wherein the synthetic nucleic acid polynucleotide primer comprises:
- (i) sufficient nucleotide residues to base pair or selectively bind and anneal to the target nucleic acid sequence under physiologic conditions, and
- (ii) one or more 3′ nucleotide residues that because of the proximity to the 3′ end of the synthetic nucleic acid polynucleotide primer are not cleaved by an enzyme;
- (c) providing or having provided a DNA polymerase, and an exonuclease or endonuclease capable of recognizing one or more 3′ nucleotide residues of the synthetic nucleic acid polynucleotide primer, and based on the recognizing the exonuclease or endonuclease cleaves the double-stranded oligonucleotide;
- (d) contacting the synthetic nucleic acid polynucleotide to the sample under conditions wherein the synthetic nucleic acid polynucleotide is capable of annealing to the target nucleic acid;
- (e) extending the length of the synthetic nucleic acid polynucleotide primer in a 5′ to 3′ direction using the DNA polymerase using the target nucleic acid as a template, thereby generating an extended synthetic nucleic acid polynucleotide primer;
- (f) contacting the double-stranded oligonucleotide with the exonuclease or endonuclease, thereby cleaving the sequence on the extended synthetic nucleic acid polynucleotide primer that was not initially recognized or cleavable by the endonuclease or exonuclease, thereby generating one or more fragments of single-stranded nucleotide,
- wherein the generation or presence the one or more fragments of single stranded-nucleotide indicates the presence of the target nucleic acid.
7 more elements
A method for detecting a target nucleic acid sequence, comprising:
- (a) providing or having provided a sample comprising a target nucleic acid;
- (b) providing or having provided a synthetic nucleic acid polynucleotide primer,
- wherein the synthetic nucleic acid polynucleotide primer comprises:
- (i) sufficient nucleotide residues to base pair and selectively bind or anneal to the target nucleic acid sequence under physiologic conditions, and
- (ii) one or more 3′ nucleotide residues that because of the proximity to the 3′ end of the synthetic nucleic acid polynucleotide primer are not cleaved by an enzyme;
- (c) providing or having provided a DNA polymerase, and an exonuclease or endonuclease capable of recognizing one or more 3′ nucleotide residues of the synthetic nucleic acid polynucleotide primer, and based on the recognizing the exonuclease or endonuclease cleaves the double-stranded oligonucleotide;
- (d) contacting the synthetic nucleic acid polynucleotide to the sample under conditions wherein the synthetic nucleic acid polynucleotide is capable of annealing to the target nucleic acid;
- (e) extending the length of the synthetic nucleic acid polynucleotide primer in a 5′ to 3′ direction using the DNA polymerase using the target nucleic acid as a template, thereby generating an extended synthetic nucleic acid polynucleotide primer;
- (f) contacting the double-stranded oligonucleotide with the exonuclease or endonuclease, thereby cleaving the sequence on the extended synthetic nucleic acid polynucleotide primer that was not initially recognized or cleavable by the endonuclease or exonuclease, thereby generating one or more fragments of single-stranded nucleotide,
- wherein the annealing and extension extending reaction conditions comprise a temperature where the cleaved and extended synthetic nucleic acid polynucleotide primer is displaced from the target nucleic acid by an unextended and uncleaved synthetic nucleic acid polynucleotide primer, in other words, the annealing and extension occurs at a temperature that thermodynamically favors binding of the uncleaved and unextended nucleic acid primer over the cleaved and extended primer, and the newly annealed uncleaved and unextended nucleic acid primer is extended in a 5′ to 3′ direction using the DNA polymerase using the target nucleic acid as a template, and again this extended nucleic acid primer can now be cleaved by the enzyme, thus generating additional cleaved primer fragments both 5′ and 3′ fragments,
- wherein the generation or presence the one or more 5′ and/or 3′ fragments of single-stranded nucleotide indicates the presence of the target nucleic acid.
8 more elements
Dependencies are inferred from the claim text.