Nucleic acid ligation systems and methods

• US 9,506,113 B2
• Filed: 12/28/2012
• Issued: 11/29/2016
• Est. Priority Date: 12/28/2011
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Phillipson, Curtis, Eshoo, Mark W., Picuri, John, Motley, Stanley T.
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US14/369,641
• Publication Number:
  US 20150004604A1
• Time in Patent Office: 1,432 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

The present disclosure relates to systems and methods for nucleic acid ligation. In particular, the present disclosure provides oligonucleotide adaptors for use in nucleic acid ligation reactions.

Claims

1. A method for analyzing nucleic acids, the method comprising:

a) contacting one or more double stranded nucleic acid inserts comprising single stranded nucleotide overhangs with a plurality of double stranded nucleic acid adaptors, wherein each of said plurality of adaptors have a distinct single stranded adaptor overhang relative to the other adaptors, wherein said adaptor overhangs have random nucleotide sequences;

b) ligating said adaptors to said one or more double stranded nucleic acid inserts with a ligase enzyme to generate ligated inserts; and

c) analyzing said ligated inserts by sequencing said ligated inserts using a next-generation sequencing technology selected from the group consisting of pyrosequencing, sequencing-by-ligation, sequence-by-synthesis (SBS), nanopore sequencing, and oligonucleotide extension.

2. The method of claim 1, wherein said plurality of adaptors comprise an adaptor overhang of 1 and/or 2 nucleotides.

3. The method of claim 1, wherein said adaptor overhangs have a plurality of distinct sequences that hybridize to said nucleotide overhangs of said inserts.

4. The method of claim 1, wherein said adaptors have single nucleotide overhangs at the 3′

and/or 5′

ends.

5. The method of claim 1, wherein two or more adaptors comprise adaptor overhangs that are different and are selected from the group consisting of a, t, c, and g.

6. The method of claim 1, wherein said adaptors have double nucleotide overhangs at the 3′

and/or 5′

ends.

7. The method of claim 1, wherein two or more adaptors comprise adaptor overhangs that are different and are selected from the group consisting of aa, ag, at, ac, ga, gg, gc, gt, tt, tc, ta, tg, cc, ct, ca, and cg.

8. The method of claim 1, wherein said plurality of adaptors comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all 16 distinct adaptor overhangs.

9. The method of claim 1, wherein said one or more double stranded nucleic acid inserts comprise distinct nucleic acid sequences.

10. The method of claim 1, wherein said single stranded nucleotide overhangs of said one or more double stranded nucleic acid inserts have a plurality of distinct sequences.

11. The method of claim 1, wherein said single stranded nucleotide overhangs of said one or more double stranded nucleic acid inserts comprise overhangs of 1 and/or 2 nucleotides.

12. The method of claim 1 wherein the plurality of double stranded nucleic acid adaptors comprises:

a) a first double stranded nucleic acid adaptor comprising an adaptor overhang of 1 or 2 nucleotides;

b) a second double stranded nucleic acid adaptor comprising an adaptor overhang of or 2 nucleotides; and

c) a third double stranded nucleic acid adaptor comprising an adaptor overhang of 1 or 2 nucleotides,

wherein;

i) the adaptor overhang of the first double stranded nucleic acid adaptor is different than the adaptor overhang of the second double stranded nucleic acid adaptor; and

ii) each of the adaptor overhangs of the first and second double stranded nucleic acid adaptors is different than the adaptor overhang of the third double stranded nucleic acid adaptor.

13. The method of claim 1 wherein each adaptor of the plurality of double stranded nucleic acid adaptors comprises a detection label.

14. The method of claim 1 wherein the plurality of adaptors comprises an adaptor overhang comprising c or g.