Solid phase methods for amplifying multiple nucleic acids
AssigneeMosaic Technologies, Inc.
InventorsT. Christian Boles, Ezra S. Abrams
Life of this patent
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Abstract
A multi-stage bridge amplification method which uses a recovered single-stranded amplification single-stranded nucleic acid molecule to initiate second and subsequent stages of bridge amplification is described.
Claims32
A solid-phase, multi-stage method of amplifying one, or more, target nucleic acid molecules comprising two or more stages of bridge amplification, wherein single-stranded amplification molecules produced in the first stage of bridge amplification initiate a second stage of bridge amplification, and each subsequent stage of bridge amplification is initiated with single-stranded amplification molecules produced in the previous stage of bridge amplification, wherein each stage of bridge amplification comprises the steps of:
- (a) hybridizing target nucleic acid molecules to a set of oligonucleotide primers immobilized on a solid support;
- (b) thermocycling to amplify the target nucleic acid molecules hybridized to the oligonucleotide primers by the formation of bridge amplification double-stranded molecules;
- (c) cleaving and denaturing the bridge amplification double-stranded molecules to form single-stranded amplification molecules;
- (d) recovering the single-stranded amplification molecules of step (c);
- (e) contacting the single-stranded amplification molecules with a fresh solid support;
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- (f) repeating steps (a) through (e) to amplify the target nucleic acid molecules.
A method of amplifying one, or more, target nucleic acid molecules using a solid support comprising two or more immobilized oligonucleotide primers, of which at least one primer specifically hybridizes to the target nucleic acid molecules, comprising the steps of:
- (a) hybridizing target nucleic acid molecules to a set of oligonucleotide primers immobilized on a solid support;
- (b) thermocycling to amplify the target nucleic acid molecules hybridized to the oligonucleotide primers forming a double-stranded amplification molecule;
- (c) cleaving and denaturing the double-stranded amplification molecule to form single-stranded amplification molecules;
- (d) recovering the single-stranded amplification molecules of step (c),
- (e) contacting the single-stranded amplification molecules with a fresh solid support, and
- (f) repeating steps (a) through (e) to amplify the target nucleic acid molecules.
A method of amplifying one, or more, target nucleic acid molecules using a solid support comprising two or more immobilized oligonucleotide primers, of which at least one specifically hybridizes to the target nucleic acid molecule, comprising the steps of:
- (a) forming a hybridization complex comprising a single-stranded target nucleic acid molecule hybridized to a first oligonucleotide primer immobilized to the solid support by contacting the target nucleic acid molecule with the solid support, under conditions suitable for hybridization;
- (b) forming a first double-stranded amplification molecule comprising the target nucleic acid molecule hybridized to a first single-stranded amplification molecule by contacting the hybridization complex of step (a) with amplification reagents under conditions suitable for a primer-mediated polymerase extension reaction, wherein a first single-stranded amplification molecule is formed by extending the first primer with deoxynucleotides to form a complementary strand to the target nucleic acid molecule;
- (c) denaturing the first double-stranded amplification molecule of (b), thereby releasing the single-stranded target molecule from the first double-stranded amplification molecule;
- (d) forming a first bridge hybridization complex comprising the first single-stranded amplification molecule of step (b) and a second oligonucleotide primer immobilized to the solid support, under conditions suitable for hybridization;
- (e) forming a second double-stranded amplification molecule by contacting the bridge hybridization complex of step (d) with amplification reagents, under conditions suitable for a primer-mediated amplification reaction, wherein a second single-stranded amplification molecule is formed that is complementary to and base-paired with the first single-stranded amplification molecule of step (d);
- (f) denaturing the second double-stranded amplification molecule of (e) to produce first and second single-stranded amplification molecules immobilized to the solid support;
- (g) forming second bridge hybridization complexes comprising
- (i) the first single-stranded amplification molecule of step (f) and a second oligonucleotide primer immobilized to the solid support, under conditions suitable for hybridization, and
- (ii) the second single-stranded amplification molecule of step (f) and a first oligonucleotide primer immobilized to the solid support, under conditions suitable for hybridization;
- (h) forming a third and fourth double-stranded amplification molecule by contacting the second bridge hybridization complexes of step (g) with amplification reagents, under conditions suitable for a primer-mediated amplification reaction, wherein
- (i) the third double-stranded amplification molecule comprises a nascent third single-stranded amplification molecule that is complementary to and is base-paired with the first single-stranded amplification molecule, and
- (ii) the fourth double-stranded amplification molecule comprises a nascent fourth single-stranded amplification molecule that is complementary to and is base-paired with the second single-stranded amplification molecule;
- (i) cleaving one, or more, first oligonucleotide primers, thereby cleaving the third and fourth double-stranded amplification molecules from at least one attachment to the solid support, or alternatively cleaving one, or more, second oligonucleotide primers, thereby cleaving the third and fourth double-stranded amplification molecules from at least one attachment to the solid support;
- (j) denaturing the cleaved amplification double-stranded molecules of (h), thereby releasing amplification single-stranded molecules from the solid support;
- (k) applying released single-stranded molecules from step (j) to fresh solid supports comprising unused immobilized oligonucleotide primers, and
- (l) repeating steps (a) through (k) one, or more, times, thereby amplifying the target nucleic acid molecules.
A solid-phase, multi-stage method of detecting the presence or absence of one, or more target nucleic acid molecules comprising two or more stages of bridge amplification, wherein single-stranded amplification molecules produced in the first stage of bridge amplification initiate a second stage of bridge amplification, and each subsequent stage of bridge amplification is initiated with single-stranded amplification molecules produced in the previous stage of bridge amplification, wherein each stage of bridge amplification comprises the steps of:
- (a) hybridizing target nucleic acid molecules to a set of oligonucleotide primers immobilized on a solid support;
- (b) thermocycling to amplify the target nucleic acid molecules hybridized to the oligonucleotide primers by the formation of bridge amplification double-stranded molecules;
- (c) cleaving and denaturing the bridge amplification double-stranded molecules to form single-stranded amplification molecules;
- (d) recovering the single-stranded amplification molecules of step (c), and
- (e) contacting the single-stranded amplification molecules with a fresh solid support;
- (f) repeating steps (a) through (e) to amplify the target nucleic acid molecules, and
- (g) detecting the presence of the target molecules, wherein the detection of the single-stranded or double-stranded amplification molecules is indicative of the target molecules in a test sample.
A method of detecting the presence or absence of one, or more, target nucleic acid molecules using a solid support comprising two or more immobilized oligonucleotide primers, of which at least one primer specifically hybridizes to the target nucleic acid molecules, comprising the steps of:
- (a) hybridizing target nucleic acid molecules to a set of oligonucleotide primers immobilized on a solid support;
- (b) thermocycling to amplify the target nucleic acid molecules hybridized to the oligonucleotide primers forming a double-stranded amplification molecule;
- (c) cleaving and denaturing the double-stranded amplification molecule to form single-stranded amplification molecules;
- (d) recovering the single-stranded amplification molecules of step (c),
- (e) contacting the single-stranded amplification molecules with a fresh solid support, and
- (f) repeating steps (a) through (e) to amplify the target nucleic acid molecules, and
- (g) detecting the presence of the target molecules, wherein the detection of the single-stranded or double-stranded amplification molecules is indicative of the target molecules in a test sample.
Dependencies are inferred from the claim text.