Methods for controlling amplification

• US 20090286286A1
• Filed: 11/04/2008
• Published: 11/19/2009
• Est. Priority Date: 11/06/2007
• Status: Abandoned Application

• Current Assignee: Ambergen Inc.
• Original Assignee: Ambergen Inc.
• Inventors: Rothschild, Kenneth J., Lim, Mark J.
• Application Number:
  US12/290,813
• Publication Number:
  US 20090286286A1
• Time in Patent Office: Days
• US Class Current: 435/91.200
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Methods of amplifying nucleic acid on a solid support are described. Beads and template, each in known concentrations, are employed so a range of template to bead ratios can be exploited. Where the beads contain primers, the template can be amplified. After amplification, non-covalently bound template is removed, so as to leave beads with extended primers (or beads with primers that were not extended).

Claims

1. A method of amplifying nucleic acid on a solid support, comprising:

(a) providing i) a population of beads, each bead comprising one or more amplification primers, ii) a solution of amplification reagents comprising a thermostable polymerase, and iii) a population of nucleic acid template molecules,

(b) mixing said beads and said template molecules in a first aliquot of said solution of amplification reagents so as to create a mixture;

(c) treating the mixture under conditions such that at least a portion of said template molecules non-covalently bind to at least a portion of said beads to create bound template, and at least a portion of said primers on at least a portion of said beads are extended by said polymerase, so as to create treated beads;

(d) manipulating said treated beads so as to remove at least a portion of said bound template so as to create manipulated beads; and

(e) contacting said manipulated beads with a second aliquot of said solution of amplification reagents under conditions such that at least a portion of said extended primers is amplified to create loaded beads comprising immobilized amplified nucleic acid and unloaded beads lacking amplified nucleic acid.

2. The method of claim 1, wherein said manipulating of step d) comprises washing said treated beads with a denaturing solution.

3. The method of claim 2, wherein said denaturing solution comprises NaOH.

4. The method of claim 3, wherein prior to step d) between 1 and 10 primers per bead are extended.

5. The method of claim 3, wherein prior to step d) some beads comprise no extended primers.

6. The method of claim 1, wherein at step a) a known concentration of beads is provided.

7. The method of claim 6, wherein at step a) a known concentration of nucleic acid template molecules is provided.

8. The method of claim 7, wherein at step b) the number of template molecules to beads is less than one.

9. The method of claim 8, wherein at step c) fewer than 50% of the beads comprise non-covalently bound template.

10. The method of claim 1, wherein the amplification primers comprise a sequence which provides a code.

11. The method of claim 10, wherein said code identifies the origin of the nucleic acid templates.

12. The method of claim 11, wherein the origin of the nucleic acid template is a patient and the code identifies the patient.

13. The method of claim 10, wherein said code identifies the bead.

14. The method of claim 1, wherein each bead of step (a) comprises a forward and a reverse PCR primer.

15. A method of amplifying nucleic acid on a solid support, comprising:

a) providing i) a population of beads, each bead comprising forward and reverse PCR primers primers, ii) a solution of amplification reagents comprising a thermostable polymerase, and iii) a population of nucleic acid template molecules,

b) mixing said beads and said template molecules in a first aliquot of said solution of amplification reagents so as to create a mixture;

c) treating the mixture under conditions such that at least a portion of said template molecules non-covalently bind to at least a portion of said beads to create bound template, and at least a portion of said primers on at least a portion of said beads are extended by said polymerase, so as to create treated beads;

d) washing said treated beads with a denaturing solution so as to create manipulated beads; and

e) contacting said manipulated beads with a second aliquot of said solution of amplification reagents under conditions such that at least a portion of said extended primers is amplified to create loaded beads comprising immobilized amplified nucleic acid and unloaded beads lacking amplified nucleic acid.

16. The method of claim 15, further comprising:

(f) treating said immobilized amplified nucleic acid so as to release at least a portion from said loaded beads so as to create free amplified nucleic acid.

17. The method of claim 15, further comprising:

(f) transferring at least a portion of said immobilized amplified nucleic acid to a non-bead solid support.

18. The method of claim 15, wherein prior to step (a) said forward and reverse PCR primers comprised 5′

amine modifications and were attached to agarose beads comprising a plurality of primary amine reactive functional groups.

19. The method of claim 15, wherein said forward and reverse PCR primers have a region that is completely complementary to a portion of the APC gene segment 3.

20. The method of claim 15, wherein said forward primer comprises a portion encoding an N-terminal epitope tag and said reverse primer comprises a portion encoding a C-terminal epitope tag.

21. The method of claim 15, wherein said mixture is created under the conditions such that the ratio of the number of nucleic acid template molecules to the number of beads is between 0.1:

1 and 2;

1.

22. The method of claim 15, wherein said mixture is created under the conditions such that the ratio of the number of nucleic acid template molecules to the number of beads is between 2:

1 and 500, 000;

1.

23. The method of claim 22, wherein the ratio of the number of nucleic acid template molecules to the number of beads is between 1000:

1 and 100, 000;

1.

24. The method of claim 22, wherein the ratio of the number of nucleic acid template molecules to the number of beads is between 10,000:

1 and 100, 000;

1

25. The method of claim 22, wherein ratio of the number of nucleic acid template molecules to the number of beads is between 1000:

1 and 10,000;

1.

26. The method of claim 15, wherein the percentage of unloaded beads is between approximately 50% and 95%, as measured by fluorescence.

27. The method of claim 15, wherein the percentage of loaded beads is between approximately 1% and 5%, as measured by fluorescence.

28. The method of claim 15, wherein the percentage of loaded beads is between approximately 5% and 50%, as measured by fluorescence.

29. A method of amplifying nucleic acid on a solid support, comprising:

a) providing i) a population of a known concentration of beads, each bead comprising one or more amplification primers, ii) a solution of amplification reagents comprising a thermostable polymerase, and iii) a population of a known concentration of nucleic acid template molecules;

b) mixing said beads and said template molecules in a first aliquot of said solution of amplification reagents so as to create a mixture under the conditions such that the ratio of the number of nucleic acid template molecules to the number of beads is between 1;

1 and 10,000;

1;

c) treating the mixture under conditions such that at least a portion of said template molecules non-covalently bind to at least a portion of said beads to create bound template, and at least a portion of said primers on at least a portion of said beads are extended by said polymerase, so as to create treated beads;

d) manipulating said treated beads so as to remove at least a portion of said bound template so as to create manipulated beads; and

e) contacting said manipulated beads with a second aliquot of said solution of amplification reagents under conditions such that at least a portion of said extended primers is amplified to create loaded beads comprising immobilized amplified nucleic acid and unloaded beads lacking amplified nucleic acid.

30. The method of claim 29, wherein each bead of step (a) comprises a forward and a reverse PCR primer.

31. The method of claim 29, wherein said manipulating comprises washing said treated beads with a denaturing solution

32. The method of claim 31, wherein said denaturing solution comprises NaOH.

33. The method of claim 29, wherein said washing removes the majority of said non-covalently bound template.

34. The method of claim 29, further comprising:

(f) treating said immobilized amplified nucleic acid so as to release at least a portion from said loaded beads so as to create free amplified nucleic acid.

35. The method of claim 29, further comprising:

(f) transferring at least a portion of said immobilized amplified nucleic acid to a non-bead solid support.

36. The method of claim 30, wherein prior to step (a) said forward and reverse PCR primers comprised 5′

amine modifications and were attached to agarose beads comprising a plurality of primary amine reactive functional groups.

37. The method of claim 30, wherein said forward and reverse PCR primers have a region that is completely complementary to a portion of the APC gene segment 3.

38. The method of claim 30, wherein said forward primer comprises a portion encoding an N-terminal epitope tag and said reverse primer comprises a portion encoding a C-terminal epitope tag.

39. The method of claim 29, wherein the ratio of the number of nucleic acid template molecules to the number of beads is between 1:

1 and 10;

1.

40. The method of claim 29, wherein the ratio of the number of nucleic acid template molecules to the number of beads is between 10:

1 and 100;

1

41. The method of claim 29, wherein ratio of the number of nucleic acid template molecules to the number of beads is between 100:

1 and 1,000;

1.

42. The method of claim 29 wherein the percentage of unloaded beads is between approximately 50% and 95%, as measured by fluorescence.

43. The method of claim 29 wherein the percentage of loaded beads is between approximately 1% and 5%, as measured by fluorescence.

44. The method of claim 29 wherein the percentage of loaded beads is between approximately 5% and 50%, as measured by fluorescence.

45. A method of amplifying nucleic acid on a solid support, comprising:

a) providing i) a population of a known concentration of beads, each bead comprising one or more amplification primers, ii) a solution of amplification reagents comprising a thermostable polymerase, and iii) a population of a known concentration of nucleic acid template molecules,

b) mixing said beads and said template molecules in a first aliquot of said solution of amplification reagents so as to create a mixture under the conditions such that the ratio of the number of nucleic acid template molecules to the number of beads is between 0.1;

1 and 2;

1;

c) treating the mixture under conditions such that at least a portion of said template molecules non-covalently bind to at least a portion of said beads to create bound template, and at least a portion of said primers on at least a portion of said beads are extended by said polymerase, so as to create treated beads;

d) exposing said treated beads to a denaturing solution so as to create manipulated beads; and

e) contacting said manipulated beads with a second aliquot of said solution of amplification reagents under conditions such that at least a portion of said extended primers is amplified to create loaded beads comprising immobilized amplified nucleic acid and unloaded beads lacking amplified nucleic acid.

46. The method of claim 45, wherein each bead of step (a) comprises a forward and a reverse PCR primer.

47. The method of claim 45, wherein said denaturing solution comprises NaOH and said exposing comprises at least two washings of the beads.

48. The method of claim 47, wherein said washings remove at least a portion of said non-covalently bound template.

49. The method of claim 47, wherein said washings removes the majority of said non-covalently bound template.

50. The method of claim 45, further comprising:

(f) treating said immobilized amplified nucleic acid so as to release at least a portion from said loaded beads so as to create free amplified nucleic acid.

51. The method of claim 45, further comprising:

(f) transferring at least a portion of said immobilized amplified nucleic acid to a non-bead solid support.

52. The method of claim 45 wherein the percentage of unloaded beads is between approximately 50% and 99%, as measured by fluorescence.

53. The method of claim 45 wherein the percentage of loaded beads is between approximately 0.1% and 2%, as measured by fluorescence.