Methods for sequencing nucleic acids
View Patent ↗Provided herein are methods and compositions for the sequencing of long nucleic acids, such as DNA. The methods and compositions are suited for the spatial labeling and sequencing of long nucleic acid molecules.
1. A method for attaching a plurality of nucleic acids, said method comprising:
(a) stretching a target nucleic acid to be sequenced,
(b) fragmenting the target nucleic acid into a plurality of nucleic acids,
(c) incubating a substrate surface comprising a plurality of oligonucleotides attached to said substrate surface with a topoisomerase I enzyme, wherein each of said plurality of oligonucleotides comprises a duplex comprising a first adaptor, a variable region, and a second adaptor, wherein said variable region is in-between said first adaptor and said second adaptor, wherein said first adaptor is attached to said substrate surface, and wherein said second adaptor comprises a first recognition sequence of said topoisomerase I enzyme within one strand of said duplex and a second recognition sequence of said topoisomerase I enzyme at a 3′ terminal end on an opposing strand of said duplex, wherein said incubating with said topoisomerase I enzyme cleaves both strands of each of said plurality of oligonucleotides at a junction of said first and second recognition sequences and bonds said topoisomerase I enzyme to each of said plurality of oligonucleotides, thereby generating a substrate surface comprising topoisomerase I enzyme bonded to each of said plurality of oligonucleotides attached to said substrate surface; and
(d) incubating the plurality of nucleic acids with said substrate surface comprising topoisomerase I enzyme bonded to each of said plurality of oligonucleotides attached to said substrate surface, wherein said topoisomerase I enzyme bonded to each of said plurality of oligonucleotides ligates each end of each of said plurality of nucleic acids to one of said plurality of oligonucleotides attached to said substrate surface, thereby attaching said plurality of nucleic acids.
2. The method of claim 1 , wherein said topoisomerase I enzyme is from Vaccinia virus.
3. The method of claim 2 , wherein said first recognition sequence, said second recognition sequence, or both is 5′-TCCTT-3′.
4. The method of claim 2 , wherein said first recognition sequence, said second recognition sequence, or both is 5′-CCCTT-3′.
5. The method of claim 1 , wherein said stretching is performed on an immobilization substrate surface, wherein said immobilization substrate surface is different from said substrate surface comprising said plurality of oligonucleotides.
6. The method of claim 1 , wherein said stretching is performed by transfer printing.
7. The method of claim 1 , wherein said variable region comprises a barcode.
8. The method of claim 1 , wherein said first adaptor comprises a recognition sequence for a restriction enzyme.