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US 7,622,281 B2Granted
Methods and compositions for clonal amplification of nucleic acid
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsMostafa Ronaghi, Foad Mashayekhi
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Abstract
Described herein are methods and compositions relating to amplifying nucleic acid. In certain embodiments, the invention provides methods for labeling and amplifying a nucleic acid molecule.
Claims19
A method of labeling and amplifying a nucleic acid molecule in a plurality of nucleic acid molecules, comprising the steps of:
- (a) attaching a unique antiprimer P1 to each of a plurality of nucleic acid molecules to be amplified;
- (b) hybridizing a molecule from step (a) to a solid surface in a plurality of solid surfaces, wherein each of said solid surfaces is attached to a single molecule of primer P1, which is complementary to the unique antiprimer P1, to form a single captured DNA fragment on each solid surface;
- (c) extending P1 hybridized in step (b) to form a double stranded fragment, then denaturing the double stranded fragment to form a single stranded captured DNA fragment attached to each solid surface through primer P1;
- (d) labeling the single stranded captured DNA fragment from step (c) by nucleotide extension;
- (e) isolating the captured DNA fragment labeled in step (d) by the label;
- (f) attaching additional primers P1 to the solid surface after isolating the labeled, captured DNA fragment from step (e);
- and
- (g) amplifying the captured DNA fragment from step (e) using the additional primers attached in step (f).
5 more elements
A method of clonal amplification of a nucleic acid molecule, comprising the steps of:
- (a) labeling the nucleic acid molecule with a unique anti primer P1;
- (b) hybridizing said nucleic acid molecule containing antiprimer P1 to one primer P1 molecule, where only one primer P1 molecule is immobilized on a solid surface;
- (c) incubating the solid surface of (b) comprising P1 and nucleic acid molecule containing antiprimer P1 with polymerase and nucleotides under conditions permitting extension of said one molecule of immobilized primer P1 to form a double stranded fragment complementary to the nucleic acid molecule containing antiprimer P1, then denaturing the double stranded fragment to form a single stranded captured DNA fragment attached to each solid surface through primer P1;
- (d) labeling the single stranded captured DNA fragment from step (c);
- (e) isolating a solid surface containing labeled product from step (d) from solid surfaces not containing labeled product;
- (f) incubating the isolated solid surface of (e) under conditions permitting denaturing of the nucleic acid molecules containing antiprimers P1 from the extended primers P1;
- removing supernatant containing extended anti-primers as a result of the denaturing;
- incubating the solid surface under conditions permitting immobilizing of additional primers P1 on the solid surface;
- and
- (g) incubating the solid surfaces of (f) with polymerase, nucleotides, and one or more oligonucleotides that hybridize to the immobilized anti-primers of (f), under conditions permitting extension of said hybridized oligonucleotides, wherein the nucleic acid molecule is thereby amplified.
7 more elements
Dependencies are inferred from the claim text.