Systems and methods for nucleic acid sequencing

• US 9,797,010 B2
• Filed: 12/19/2008
• Issued: 10/24/2017
• Est. Priority Date: 12/21/2007
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Weitz, David A., Agresti, Jeremy, Weiner, Michael P., Abate, Adam R., Hung, Tony
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/809,120
• Publication Number:
  US 20110267457A1
• Time in Patent Office: 3,231 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads

Abstract

The present invention relates to systems and methods for sequencing nucleic acids, including sequencing nucleic acids in fluidic droplets. In one set of embodiments, the method employs sequencing by hybridization using droplets such as microfluidic droplets. In some embodiments, droplets are formed which include a target nucleic acid, a nucleic acid probe, and at least one identification element, such as a fluorescent particle. The nucleic acid probes that hybridize to the target nucleic acid are determined, in some instances, by determining the at least one identification element. The nucleic acid probes that hybridize to the target nucleic acid may be used to determine the sequence of the target nucleic acid. In certain instances, the microfluidic droplets are provided with reagents that modify the nucleic acid probe. In some cases, a droplet, such as those described above, is deformed such that the components of the droplets individually pass a target area.

Claims

1. A method, comprising:

providing a first microfluidic droplet containing a nucleic acid probe and at least three distinguishable identification elements, wherein each of the at least three distinguishable identification elements is associated with a different oligonucleotide sequence comprising at least one universal nucleic acid residue;

providing a second microfluidic droplet comprising a target nucleic acid; and

fusing at least some of the fluid in the first fluidic droplet and at least some of the fluid in the second microfluidic droplet to form a fused droplet.

2. The method of claim 1, comprising determining a sequence of at least a portion of the target nucleic acid.

3. The method of claim 1, further comprising determining at least a portion of the target nucleic acid by determining at least one of the at least three identification elements contained within the fused droplet.

4. The method of claim 1, further comprising determining at least one of the at least three identification elements.

5. The method of claim 1, wherein the nucleic acid probe contains at least four residues.

6. The method of claim 1, wherein the nucleic acid probe contains at least one locked nucleic acid residue.

7. The method of claim 1, further comprising determining association between the target nucleic acid and the nucleic acid probe.

8. The method of claim 1, wherein the nucleic acid probe comprises a quencher.

9. The method of claim 1, wherein the droplet contains four distinguishable identification elements.

10. The method of claim 1, wherein each of the at least three distinguishable identification elements is attached to the different oligonucleotide sequence.

11. The method of claim 1, wherein the first microfluidic droplet or the second microfluidic droplet comprises a ligase.

12. The method of claim 11, further comprising ligating the nucleic acid probe to one of the different oligonucleotide sequences associated with one of the at least three distinguishable identification elements.

13. The method of claim 1, wherein each of the different oligonucleotide sequences differs by one nucleic acid residue.

14. The method of claim 13, wherein the one nucleic acid residue is at the same location on each of the different oligonucleotide sequences.

15. The method of claim 1, wherein each of the different oligonucleotide sequences has a single non-universal nucleic acid residue.

16. A method, comprising:

providing a first microfluidic droplet containing a nucleic acid probe and at least three distinguishable identification elements, wherein each distinguishable identification element is associated with a different oligonucleotide sequence;

providing a second microfluidic droplet comprising a target nucleic acid;

fusing at least some of the fluid in the first fluidic droplet and at least some of the fluid in the second microfluidic droplet to form a fused droplet; and

ligating the nucleic acid probe to one of the different oligonucleotide sequences associated with one of the at least three distinguishable identification elements.

17. The method of claim 16, comprising determining a sequence of at least a portion of the target nucleic acid.

18. The method of claim 16, further comprising determining at least a portion of the target nucleic acid by determining at least one of the at least three identification elements contained within the fused droplet.

19. The method of claim 16, wherein each of the at least three distinguishable identification elements is attached to the different oligonucleotide sequence.

20. The method of claim 16, wherein each of the different oligonucleotide sequences differs by one nucleic acid residue.