HIGH THROUGHPUT DNA SEQUENCING METHOD AND APPARATUS

• US 20100021915A1
• Filed: 12/11/2007
• Published: 01/28/2010
• Est. Priority Date: 12/11/2006
• Status: Abandoned Application

• Current Assignee: Thomas Jefferson University (Main Line Health System), UD Technology Corporation
• Original Assignee: Thomas Jefferson University (Main Line Health System), UD Technology Corporation
• Inventors: Khan, Rishi Lee, Schwaber, James Stephen
• Application Number:
  US12/518,332
• Publication Number:
  US 20100021915A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/619 being a video camera

Abstract

The present invention relates to a method for high throughput nucleic acid sequencing using a multi-bead flow cell and pyrophosphate sequencing, a sequencer capable of performing this method, and a kit of the pyrophosphate sequencing reagents.

Claims

1. A method for sequencing a nucleic acid molecule, comprising:

(a) providing a sequencer, comprising;

(i) a reservoir; and

,

(ii) a microfluidic flow cell, comprising;

a flow chamber, comprising;

a planar imagable area; and

,

a plurality of beads immobilized onto the planar imagable area;

wherein;

the plurality of reservoirs is fluidly connected to the flow cell; and

,

a substantial portion of the beads, further comprise;

a plurality of nucleic acid primers attached thereto, wherein the nucleic acid primers present on an individual bead are homogeneous;

(b) contacting the target nucleic acid molecule with said beads and pyrophosphate sequencing reagents, comprising;

a nucleotide triphosphate;

a polymerase;

a pyrophosphate to ATP converting enzyme; and

,

an ATP detecting enzyme; and

(c) detecting the resulting optical signals, wherein each optical signal is indicative of a reaction of pyrophosphate sequencing reagents with a target nucleic acid molecule on a bead, thereby sequencing the nucleic acid.

2. The method of claim 1, wherein the microfluidic flow cell further comprises:

a first fluid inlet fluidly connected to the flow chamber and fluidly connected to the plurality of reservoirs; and

,

a first fluid outlet fluidly connected to the flow chamber.

3. The method of claim 1, wherein the first fluid inlet and first fluid outlet are connected to the same surface of the flow cell and are separated by the imagable area.

4. The method of claim 1, wherein the planar imagable area comprises glass.

5. The method of claim 1, wherein the space between the imagable area and the wall of the flow cell is from 5 μ

m-100 μ

m.

6. The method of claim 1, wherein the contacting is performed by delivering the pyrophosphate sequencing reagents from the plurality of reservoirs to the flow chamber whereby the nucleic acids are exposed to the reagents.

7. The method of claim 6, wherein the contacting further comprises sequential delivery of homogeneous nucleotide triphosphates.

8. The method of claim 1, wherein the pyrophosphate sequencing byproduct is detected by contacting it with an ATP sulfurylase under conditions that allow for formation of ATP.

9. The method of claim 8, wherein the ATP sulfurylase is a thermostable ATP sulfurylase.

10. The method of claim 1, wherein the pyrophosphate sequencing byproduct is detected by contacting it with a pyruvate orthophosphate dikinase under conditions that allow for formation of ATP.

11. The method of claim 10, wherein the pyruvate is a thermostable pyruvate orthophosphate dikinase.

12. The method of claim 1, further comprising washing the flow cell with a wash buffer between each delivery of a nucleotide triphosphate.

13. The method of claim 1, wherein the nucleic acid molecules are attached to the beads via their 5′

ends via a biotin-streptavidin binding linkage.

14. The method of claim 1, wherein the beads are immobilized onto the imagable surface via a binding pair or a chemical bond.

15. The method of claim 4, wherein the beads are immobilized to the imagable glass surface via a strepavadin-biotin-protein-silanyl linkage between nucleic acid bound to the bead and the imagable glass.

16. The method of claim 4, wherein the beads are immobilized to the imagable glass surface via a 3′

nucleic acid comprising a primary amine group-silanyl linkage.

17. The method of claim 1, wherein the diameter of the beads is from 1 μ

m-20 μ

m.

18. The method of claim 1, wherein the beads are packed such that the free space is between the beads is less than 20% of the total imagable area.

19. The method of claim 1, wherein the ATP detecting enzyme is luciferase, which produces light for detection.

20. The method of claim 21, wherein the luciferase is a thermostable firefly luciferase.

21. The method of claim 1, wherein the signal detection is performed by a CMOS camera.

22. The method of claim 1, wherein the optical signals from the pyrophosphate sequencing reaction are imaged before the reagents and byproducts diffuse far enough away from the bead incorporating the nucleotide sequence that the light can no longer be localized to that specific bead.

23. The method of claim 1, wherein the reaction is imaged within 10 Ms-1000 Ms.

24. The method of claim 1, wherein the optical signal is light and signal deconvolution is used to localize the light signal to a bead.

25. The method of claim 1, wherein the sequencer comprises a plurality of flow cells.

26. A sequencer for sequencing a target nucleic acid molecule, comprising:

(i) a reservoir; and

,

(ii) a microfluidic flow cell, comprising;

a flow chamber comprising;

a planar imagable area; and

,

a plurality of beads immobilized onto the planar imagable area;

wherein;

the plurality of reservoirs is fluidly connected to the flow cell; and

,

a substantial portion of the beads further comprise;

a plurality of nucleic acid molecules attached thereto, wherein the nucleic acid molecules present on an individual bead are homogeneous;

27. A kit for sequencing a nucleic acid molecule, comprising:

a. a polymerase;

b. a pyrophosphate to ATP converting enzyme;

c. an ATP detecting enzyme;

d. nucleotides, or optionally nucleotide analogues, optionally including, in place of dATP, a dATP analogue which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said pyrophosphate to ATP converting enzyme;

e. optionally dideoxynucleotides, or optionally dideoxynucleotide analogues, optionally ddATP being replaced by a ddATP analogue which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said PPi-detection enzyme;

f. optionally deoxynucleotides or dideoxynucleotides capped on the 3′

side with a 2-nitrobenzyl moiety to prevent successive incorporation of nucleotide in homopolymeric regions of DNA sequence.