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US 12,110,534 B2Granted
Generation of single-stranded circular DNA templates for single molecule sequencing
AssigneesRoche Sequencing Solutions, Inc., Kapa Biosystems, Inc.
InventorsDaleen Badenhorst, Michael S. Berry, Richard Dannebaum, Ashley Hayes, Severine Margeridon, Martin Ranik, Etienne Slabbert
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Abstract
The invention is a novel method of sequencing nucleic acids involving making and sequencing a library of single stranded circular target nucleic acids.
Claims11
A method of separately sequencing each strand of a double-stranded target nucleic acid, wherein the method comprises the following steps:
- (a) in a reaction mixture, joining a double-stranded target nucleic acid to an adaptor to form an adapted target nucleic acid, wherein the adaptor comprises primer-binding sites, and wherein the adaptor does not consist of a long strand and a short strand;
- (b) amplifying the adapted target nucleic acid with a pair of primers, wherein the pair of primers are complementary to the primer-binding sites, thereby forming an amplicon, wherein one primer in the pair of primers comprises a modified nucleotide affecting a rate of digestion by an exonuclease;
- (c) contacting the reaction mixture with an exonuclease, thereby eliminating from the reaction mixture the first of the two complementary strands of the amplicon;
- (d) circularizing the second of the two complementary strands of the amplicon to form a single stranded circle;
- (e) annealing a sequencing primer to the single-stranded circle;
- and
- (f) extending the primer, thereby sequencing one strand of the target nucleic acid.
4 more elements
Dependencies are inferred from the claim text.