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US 11,981,961 B2Granted
Methods for constructing copies of nucleic acid molecules
AssigneeVastogen, Inc.
InventorDimitra Tsavachidou
Life of this patent
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Abstract
Methods for constructing consecutively connected copies of nucleic acid molecules are disclosed. Consecutively connected copies of nucleic acid molecules can be used to perform sequencing of the same nucleic acid molecules several times, improving overall accuracy of sequencing. Connected copies of nucleic acid molecules can be constructed by circularizing nucleic acid molecules, performing rolling circle amplification and debranching with nicking and polymerases comprising 5′-3′ exonuclease and/or flap endonuclease activity.
Claims4
A method comprising steps of:
- (i) producing a circularized nucleic acid molecule by attaching a nucleic acid molecule to at least one adaptor comprising at least one nicking endonuclease recognition site and circularizing the nucleic acid molecule;
- (ii) performing a rolling-circle amplification by exposing the circularized nucleic acid molecule to nucleotides, strand-displacing polymerases, and at least one primer, thereby producing one or more double-stranded nucleic acid constructs;
- (iii) exposing the one or more double-stranded nucleic acid constructs produced in step (ii) to nicking endonucleases that recognize the at least one nicking endonuclease recognition site present in the one or more double-stranded nucleic acid constructs produced in step (ii) such that at least one nick is produced on only one strand of double-stranded regions of the one or more double-stranded nucleic acid constructs produced in step (ii), thereby producing one or more nicked nucleic acid constructs;
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- (iv) exposing the one or more nicked nucleic acid constructs of step (iii) to polymerases comprising 5′-3′ exonuclease and/or flap endonuclease activity.
2 more elements
Dependencies are inferred from the claim text.