Nucleic acid library generation methods and compositions
AssigneeBio-Rad Laboratories, Inc.
InventorJohn A. Luckey
Life of this patent
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Abstract
Methods of forming a library of polynucleotide templates for DNA sequencing are disclosed. The methods include distributing a nucleic acid sample into different droplets in a set of emulsion droplets. The droplets into which the polynucleotide species are distributed include primers which binds to the polynucleotide species and are extended by a polymerase contained within the droplet. A set of extended primers are created simultaneously in a large number of droplets. An enriched set of extended primers are distributed into a second set of emulsion droplets containing a second primer which is extended by a polymerase contained within the second set of droplets.
Claims13
A method of forming a library of polynucleotide templates for sequencing, the method comprising the steps of:
- distributing a nucleic acid sample comprising a plurality of different polynucleotide species into a first set of emulsion droplets, wherein a plurality of droplets comprise a single polynucleotide species and a first primer comprising:
- (1) a region complementary to the polynucleotide species;
- (2) a region for binding to a first universal primer sequence;
- and
- (3) a purification binding moiety;
- extending the first primer, whereby a set of extended first primers are created;
- breaking the first emulsion droplets;
- creating an enriched set of extended first primers;
- distributing members of the enriched set of extended first primers into a second set of emulsion droplets, wherein a plurality of droplets comprise a single first extended primer species and a second primer comprising:
- (1) a region complementary to the extended primer;
- and
- (2) a region for binding to a second universal primer sequence;
- and
- extending the second primer in the droplets, whereby a library of polynucleotide templates comprising the first and second universal primer binding sequences are created.
Dependencies are inferred from the claim text.