Apparatus and methods for sequencing nucleic acids in microfluidic systems

• US 6,107,044 A
• Filed: 06/16/1999
• Issued: 08/22/2000
• Est. Priority Date: 10/16/1997
• Status: Expired due to Term

• Current Assignee: Caliper Technologies Corp (Revvity, Inc.)
• Original Assignee: Caliper Technologies Corp (Revvity, Inc.)
• Inventors: Nikiforov, Theo T.
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US09/334,527
• Time in Patent Office: 433 Days
• Field of Search: 435/6, 210/198.2
• US Class Current: 435/6.12
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

The present invention is generally directed to microfluidic systems and methods of using such systems in the determination of the nucleotide sequence of target nucleic acid sequences (referred to herein as the "target"). In particular, the present invention provides methods and systems for determining the relative positions within a target nucleic acid sequence that are occupied by a given nucleotide, e.g., A, T, G or C, by separating mixtures of nested sets of fragments of the target nucleic acid, which sets each include fragments that terminate in a different given nucleotide.

Claims

1. A method of mixing and separating nucleic acids, the method comprising:

mixing a plurality of sets of nested nucleic acid fragments in a mixing channel;

flowing the resulting nucleic acid fragment mixture to a fluidly coupled separated channel comprising a separation matrix; and

,

separating the nucleic acid fragment mixture by flowing the mixture through the separation channel, thereby providing separated nucleic acid fragments.

2. The method of claim 1, further comprising providing a plurality of source of the nested nucleic acid fragments, thereby providing the plurality of sets of nested nucleic acid fragments which are mixed in the mixing channel.

3. The method of claim 2, wherein the plurality of sources are fluidly coupled to the mixing channel, the method further comprising flowing the plurality of sets of nested nucleic acid fragments from the plurality of sources into the mixing channel.

4. The method of claim 1, wherein each of the sets of nested nucleic acid fragments are provided by performing a plurality of dideoxy chain termination reactions using a selected target nucleic acid as a template.

5. The method of claim 4, wherein each of the plurality of dideoxy chain reactions comprises a different dideoxy chain termination nucleotide.

6. The method of claim 1, wherein each of the sets of nested nucleic acid fragments are provided by performing an extension reaction in the presence of boronated nucleotides, using a target nucleic acid as a template.

7. The method of claim 6, wherein each of the plurality of extension reactions comprises a different boronated nucleotide.

8. The method of claim 1, wherein each of the sets of nested nucleic acid fragments are provided by performing a dideoxy chain termination reaction using a target nucleic acid as a template, or by performing an extension reaction in the presence of boronated nucleotides using the target nucleic acid as a template, wherein each of the sets of nested nucleic acid fragments comprise a first label, wherein the first label is the same for each of the sets of nested nucleic acid fragments.

9. The method of claim 1, wherein each of the sets of nested nucleic acid fragments comprise a first label, wherein the first label is the same for each of the sets of nested nucleic fragments.

10. The method of claim 1, comprising mixing the plurality of sets of nested fragments at detectably different concentrations to provide the mixture.

11. The method of claim 1, further comprising detecting the separated nucleic acid fragments.

12. The method of claim 1, wherein the plurality of sets of nested nucleic acid fragments are made by performing one or more sequencing reaction using a target nucleic acid as a template.

13. The method of claim 12, further comprising determining the sequence of the target nucleic acid by detecting the separated nucleic acid fragments.

14. The method of claim 1, further comprising detecting the separated nucleic acid fragments, thereby providing signals corresponding to the detected nucleic acid fragments and recording the presence of the signals.

15. The method of claim 14, further comprising analyzing the resulting recorded signal to distinguish between different sets of nucleic acid fragments in the plurality of sets of nucleic acid fragments.

16. The method of claim 15, wherein the analyzing step comprises determining an average peak height for a plurality of the signals.

17. The method of claim 15, wherein the analyzing step comprises determining an average peak height for a plurality of the signals and determining the relative position of a plurality of the plurality of signals.

18. The method of claim 17, wherein the sets of nested nucleic acid fragments correspond to a sequence of a target nucleic acid, the method further comprising converting the average peak height and relative position for the plurality or a plurality of signals into a nucleic sequence corresponding to the sequence of the target nucleic acid.

19. The method of claim 15, wherein the analyzing step is performed using a computer.

20. The method of claim 19, wherein the computer reports the relative position of the signals.

21. The method of claim 1, wherein the nucleic acid fragment mixture is flowing to the fluidly coupled separation channel using an electrokinetic material transport system.

22. The method of claim 1, wherein the nucleic acid fragment mixture is flowed to the fluidly coupled separation channel using an electrokinetic material transport system, wherein the mixture is introduced from the mixing channel into the separation channel by performing a pinched injection.

23. The method of claim 1, wherein the separation matrix comprises one or more of:

an acrylamide polymer, a methylacrylamide polymer, a dimethylacrylamide polymer, hydroxymethyl cellulose, and agarose.

24. The method of claim 1, further comprising providing a body structure comprising the separation and mixing channels.

25. The method of claim 24, wherein the body structure is fabricated from one or more of:

a polymer, glass, and quartz.

26. The method of claim 24, wherein the body structure is provided by fabricating channels in a first substrate corresponding to the separation and mixing channels and covering the first substrate with a cover layer.