Method and Reagent Kit for Targeted Genomic Enrichment
AssigneeAdvancedseq LLC
InventorZhaohui ZHOU
Life of this patent
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Abstract
A method for cleaving a target DNA to isolate a target DNA fragment sequence of interest, directed by a targeting oligonucleotide followed by enriching the target DNA fragment sequence is disclosed. The targeting oligonucleotide binds to the target DNA during DNA cleavage. After cleavage, the target DNA fragment sequence of interest is modified using either a ligation or a polymerase extension method. The resulting target DNA fragment sequence of interest is enriched by exonuclease treatment.
Claims20
A method for improving specificity for enrichment of a target DNA comprising:
- a. providing a genomic target DNA sample comprising one or more double-stranded (DS) target fragments within at least one target region, wherein the at least one target region comprises one or more double-stranded target DNA fragments;
- b. cleaving both ends of the one or more double-stranded target fragment(s), with a CRISPR/Cas12a-guide RNA (gRNA) endonuclease complex, wherein the endonuclease is a sequence specific DNA nuclease;
- and wherein the gRNA is complementary to each flanking region of its respective end within or outside the one or more target fragment(s) to be enriched to create cohesive 5′ over-hanging single-stranded ends;
- c. incorporating modified nucleotides (ON) using a DNA polymerase to each of the 5′ overhanging ends of the one or more DS target fragment(s), wherein the modified nucleotides protect the one or more DS target fragment(s) from exonuclease digestion;
- and
- d. digesting the unprotected genomic DNA with an exonuclease, wherein the one or more target DS fragment(s) are specifically enriched.
A kit comprising:
- a. a sequence specific DNA nuclease;
- b. a DNA polymerase;
- c. modified nucleotides;
- d. DNA exonuclease;
- and optionally
- e. a ligase.
A DNA enrichment kit comprising at least two of the following reagents:
- a. a Cas12a protein or a variant thereof;
- b. a targeting oligonucleotide (guide-RNA);
- c. a DNA polymerase;
- d. modified nucleotides;
- e. a DNA exonuclease;
- f. a DNA or RNA ligase;
- and
- g. DNA/RNA linkers.
Dependencies are inferred from the claim text.