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US 2021/0246496 A1Published application
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.
Claims20
A method of preparing a 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, c7dGTP, dCTP, dATP, dTTP, and a DNA polymerase;
- (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);
- and
- (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.
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