METHOD FOR NUCLEIC ACID ISOLATION AND AMPLIFICATION

• US 20090047674A1
• Filed: 04/04/2008
• Published: 02/19/2009
• Est. Priority Date: 08/09/2004
• Status: Active Grant

• Current Assignee: Generation Biotech, LLC, Genovision Incorporated, Qiagen GmbH (Qiagen NV)
• Original Assignee: Generation Biotech, LLC, Genovision Incorporated, Qiagen GmbH (Qiagen NV)
• Inventors: Murphy, Nancy, Dapprich, Johannes, Korfhage, Christian
• Granted Patent: US 8,465,925 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

The present invention provides methods and compositions for sequence-specific isolation of polynucleotide molecules from nucleic acid populations and subsequent amplification of isolated polynucleotide molecules or fragments thereof.

Claims

1. A method for amplifying a polynucleotide molecule of interest or a fragment thereof, comprising:

(a) isolating a polynucleotide molecule from a nucleic acid population using an immobilizable separation group to provide an isolated polynucleotide molecule, and

(b) isothermally amplifying the isolated polynucleotide molecule or a fragment thereof.

2. The method of claim 1 wherein

(A) the nucleic acid population comprises the polynucleotide molecule of interest,

(B) one strand of the polynucleotide molecule comprises a target nucleic acid sequence and a distinguishing element,

(C) the target nucleic acid sequence is within 100 nucleotides of the distinguishing element in the one strand of the polynucleotide molecules, and

(D) step (a) comprises:

(i) contacting the nucleic acid population with a targeting element that binds specifically to the target nucleic acid sequence in the polynucleotide molecule,

(ii) selectively attaching the immobilizable separation group to the targeting element bound to the target nucleic acid sequence in the polynucleotide molecule to form a targeting element-separation group complex,

(iii) immobilizing to a substrate via the separation group the targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, and

(iv) removing the immobilized targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, thereby isolating the polynucleotide molecule from the nucleic acid population.

3. The method of claim 2 wherein the polynucleotide molecule of interest is a genomic DNA molecule.

4. The method of claim 2 wherein the target nucleic acid sequence is located immediately 3′

to the distinguishing element in the one strand of the polynucleotide molecule.

5. The method of claim 2 wherein the targeting element comprises an oligonucleotide.

6. The method of claim 2 wherein the separation group comprises an immobilizable nucleotide.

7. The method of claim 6 wherein the immobilizable nucleotide is a terminating nucleotide.

8. The method of claim 6 wherein the immobilizable nucleotide is a non-terminating nucleotide.

9. The method of claim 2 wherein the distinguishing element is a polymorphic sequence.

10. The method of claim 2 wherein the distinguishing element is a single nucleotide polymorphism.

11. The method of claim 2 wherein

(1) the targeting element comprises an oligonucleotide,

(2) the separation group comprises an immobilizable nucleotide, and

(3) the separation group is attached to the targeting element by extending the oligonucleotide in the presence of the immobilizable nucleotide, thereby forming an extension product that comprises the immobilizable nucleotide.

12. The method of claim 11 wherein

(4) the 3′

terminus of the oligonucleotide is complementary to the distinguishing element or a portion thereof in the polynucleotide molecule,

(5) the immobilizable nucleotide is non-terminating, and

(6) the extension product comprises multiple separation groups.

13. The method of claim 11 wherein

(4) the target nucleic acid sequence is immediately 3′

to the distinguishing element, and

(5) the immobilizable nucleotide is terminating and complementary to the distinguishing element or a portion thereof.

14. The method of claim 1 wherein

(A) the nucleic acid population comprises the polynucleotide molecule of interest,

(B) one strand of the polynucleotide molecule comprises a target nucleic acid sequence and a distinguishing element,

(C) the target nucleic acid sequence is within 100 nucleotides of the distinguishing element in the one strand of the polynucleotide molecule, and

(D) step (a) comprises:

(i) contacting the nucleic acid population with a targeting element-separation group complex, wherein the targeting element-separation group complex binds specifically to the target nucleic acid sequence in the polynucleotide molecule,

(ii) selectively stabilizing the binding of the targeting element-separation group complex to the target nucleic acid sequence in the polynucleotide molecule,

(iii) immobilizing to a substrate via the separation group the stabilized targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, and

(iv) removing the immobilized stabilized targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, thereby isolating the polynucleotide molecule from the nucleic acid population.

15. The method of claim 2 wherein

(1) the targeting element comprises an oligonucleotide, and

(2) the 3′

terminus of the oligonucleotide is complementary to the distinguishing element or a portion thereof in the polynucleotide.

18. The method of claim 1 wherein the polynucleotide molecule is at least about 10 kb in length.

19. The method of claim 1 wherein the distinguishing element is haplotype-specific.

20. The method of claim 1 wherein the distinguishing element is locus-specific.

22. The method of claim 1 wherein step (b) amplifies a particular region of the isolated polynucleotide molecule.

23. The method of claim 22 wherein step (b) is performed in the presence of a first set of specific primers each of which is at least substantially complementary to the particular region in the strand of the polynucleotide molecule that comprises the target nucleic acid sequence.

24. The method of claim 23 wherein step (b) is performed further in the presence of a second set of specific primers each of which is at least substantially complementary to the particular region in the strand of the polynucleotide molecule that does not comprise the target nucleic acid sequence.

25. The method of claim 23 wherein the first set of specific primers are about 0.5 kb apart from their neighboring primers when annealing to the strand of the isolated polynucleotide molecule that comprises the target nucleic acid sequence.

26. The method of claim 24 wherein the second set of specific primers are about 0.5 kb apart from their neighboring primers when annealing to the strand of the isolated polynucleotide molecule that does not comprise the target nucleic acid sequence.

27. The method of claim 25 wherein step (b) is performed further in the presence of a set of random primers.

28. The method of claim 26 wherein step (b) is performed further in the presence of a set of random primers.

29. The method of claim 27 wherein the random primers are about 2 kb apart from their neighboring primers.

30. The method of claim 28 wherein the second set of random primers are about 2 kb apart from their neighboring primers.

33. The method of claim 1 wherein step (b) is performed in the presence of end-specific primers.

35. The method of claim 33 wherein step (b) is performed further in the presence of center-specific primers.

41. The method of claim 1 further comprising:

(c) characterizing one or more polymorphic sites in the amplified polynucleotide molecule or fragment thereof, and optionally

(d) assembling information of the characterized sites.

43. The method of claim 2 further comprising dissociating the isolated polynucleotide molecule from the substrate prior to step (b).

44. The method of claim 14 further comprising dissociating the isolated polynucleotide molecule from the substrate prior to step (b).

16-17. -17. (canceled)

21. (canceled)

31-32. -32. (canceled)

34. (canceled)

36-40. -40. (canceled)

42. (canceled)

45. A method for amplifying multiple polynucleotide molecules of interest from a population of nucleic acid molecules, comprising:

(a) isolating multiple polynucleotide molecules from a nucleic acid population using one or more immobilizable separation groups to provide isolated polynucleotide molecules of interest, and

(b) isothermally amplifying the isolated polynucleotide molecules or fragments thereof.

46. The method of claim 45 wherein step (a) comprises:

(A) contacting a nucleic acid population that comprises multiple polynucleotide molecules of interest with multiple targeting elements so that each targeting element binds specifically to a target nucleic acid sequence of its corresponding polynucleotide molecule, wherein

(i) the target nucleic acid sequence is located within 100 nucleotides of a distinguishing element in one strand of the polynucleotide molecule, and

(ii) the distinguishing element distinguishes the polynucleotide molecule from another nucleic acid molecule that is nearly identical to the polynucleotide molecule,

(B) selectively attaching separation groups to the multiple targeting elements bound to the target nucleic acid sequences of corresponding polynucleotide molecules to form targeting element-separation group complexes,

(C) immobilizing to substrate(s) via the separation groups the targeting element-separation group complexes to which the target nucleic acid sequences in the polynucleotide molecules are bound, and

(D) removing the immobilized targeting element-separation group molecules are bound to isolate the polynucleotide molecules from the population of nucleic acid molecules.

47. The method of claim 46 wherein different separation groups are attached to different targeting elements.

48. The method of claim 46 wherein identical separation groups are attached to different targeting elements.

49. The method of claim 45 wherein step (a) comprises:

(A) contacting a nucleic acid population that comprises multiple polynucleotide molecules of interest with multiple targeting elements to which separation groups are attached so that each targeting element binds specifically to a target nucleic acid sequence of its corresponding polynucleotide molecule, wherein

(i) the target nucleic acid sequence is located within 100 nucleotides of a distinguishing element in one strand of the polynucleotide molecule, and

(ii) the distinguishing element distinguishes the polynucleotide molecule from another nucleic acid molecule that is nearly identical to the polynucleotide molecule,

(B) selectively stabilizing the binding of the targeting elements to the target nucleic acid sequences of their corresponding polynucleotide molecules to form stabilized targeting element-separation group complexes to which the target nucleic acid sequences in the polynucleotide molecules are bound,

(C) immobilizing to substrate(s) via the separation groups the targeting element-separation group complexes to which the target nucleic acid sequences in the polynucleotide molecules are bound, and

(D) removing the immobilized targeting element-separation group complexes to which the target nucleic acid sequences in the polynucleotide molecules are bound to isolate the polynucleotide molecules from the population of nucleic acid molecules.

52. The method of claim 46 wherein at least three different polynucleotide molecules of interest are isolated.

50-51. -51. (canceled)

53. A method for amplifying a genomic DNA molecule of interest or a fragment thereof, wherein the genomic DNA molecule of interest comprises a polymorphic sequence, comprising:

(a) contacting a genomic DNA population that comprises the genomic DNA molecule of interest with an oligonucleotide, wherein

(i) the oligonucleotide comprises a sequence at least substantially complementary to a target nucleic acid sequence in one strand of the genomic DNA molecule of interest,

(ii) the target nucleic acid sequence is located immediately 3′

to the polymorphic sequence in the one strand of the genomic DNA molecule of interest, and

(iii) the 3′

portion of the oligonucleotide is complementary to the polymorphic sequence or a portion thereof when annealing to the one strand of the genomic DNA molecule of interest,

(b) extending the oligonucleotide in the presence of an immobilizable nucleotide using the one strand of the genomic DNA molecule of interest to which the oligonucleotide anneals as a template to provide an extension product,

(c) immobilizing to a substrate via the immobilizable nucleotide the extension product to which the genomic DNA molecule of interest is bound,

(d) removing the immobilized extension product to which the genomic DNA molecule of interest is bound to thereby isolate the genomic DNA molecule of interest from the genomic DNA population,

(e) optionally elute the genomic DNA molecule of interest from the substrate, and

(f) isothermally amplifying the isolated or eluted genomic DNA molecule of interest or a fragment thereof.

54. A method for assembling a haplotype comprising:

(a) providing a nucleic acid population from an organism for which a haplotype is of interest;

(b) separately isolating polynucleotide molecules by haplotype-specific extraction using multiple substrates, wherein

(i) polynucleotide molecules are isolated at multiple extraction sites using each substrate,

(ii) no polynucleotide molecules isolated at one extraction site using one substrate comprise a polymorphic site also present in polynucleotide molecules isolated at other extraction sites using the same substrate,

(iii) one or more polynucleotide molecules isolated at one extraction site using one substrate comprise a polymorphic site also present in polynucleotide molecules isolated at a neighboring extraction site using other substrates,

(c) separately characterizing polymorphic sites in polynucleotide molecules isolated using each substrate;

(d) assembling a haplotype based on the characterization of polymorphic sites present in polynucleotide molecules isolated using more than one substrate.

55. The method of claim 54 further comprising isothermally amplifying polynucleotide molecules isolated using the multiple substrates prior to step (c).