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US 12,195,791 B2Granted
Target enrichment via enzymatic digestion in next generation sequencing
AssigneeSaint Louis University
InventorsXiaofeng Fan, Adrian Michael Di Bisceglie
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Abstract
Provided herein are methods of preparing a template nucleic acid for next-generation sequencing. The methods comprise performing primer extension using c7dGTP instead of dGTP, which then allows for the input nucleic acids to be selectively digested by c7dGTP-resistant restriction enzymes, thereby enriching the template nucleic acid prior to next-generation sequencing library preparation.
Claims18
A method of preparing a target enriched nucleic acid template for next-generation sequencing, the method comprising:
- (a) incubating a population of nucleic acid molecules comprising the template nucleic acid with a primer that hybridizes to the template nucleic acid, c7dGTP, dCTP, dATP, dTTP, and a DNA polymerase under conditions to allow for primer extension;
- (b) incubating the product of step (a) with a splinter oligonucleotide and T4 DNA ligase under conditions to allow for the self-ligation of the extension product of step (a);
- (c) incubating the product of step (b) with c7dGTP-resistant restriction enzymes, Exonuclease I, and Exonuclease III under conditions to allow for the digestion of non-target and background nucleic acid;
- (d) incubating the product of step (c) with a primer that hybridizes to the primer used in step (a), dNTPs, and a DNA polymerase under conditions to allow for rolling circle amplification;
- and
- (e) incubating the product of step (d) with degenerate primers, dNTPs, and a DNA polymerase under conditions to allow for rolling circle amplification, thereby generating a target enriched nucleic acid template for next-generation sequencing.
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Dependencies are inferred from the claim text.