Method for nucleic acid isolation and amplification

• US 8,465,925 B2
• Filed: 04/04/2008
• Issued: 06/18/2013
• 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: Dapprich, Johannes, Murphy, Nancy, Korfhage, Christian
• Primary Examiner(s): Kim, Young J
• Application Number:
  US12/098,197
• Publication Number:
  US 20090047674A1
• Time in Patent Office: 1,901 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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, wherein

(i) the amplification is a sequence-biased amplification which is defined as a hybrid of generic amplification and sequence-specific amplification performed in the presence of both random primers and sequence-specific primers, or

(ii) the amplification is a sequence-specific amplification performed in the presence of center-specific primers and end-specific primers.

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 2 wherein the distinguishing element is haplotype-specific.

15. The method of claim 2 wherein the distinguishing element is locus-specific.

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

17. 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.

18. The method of claim 17 wherein

(1) the targeting element of the targeting element-separation group complex comprises an oligonucleotide, and

(2) the 3′

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

19. The method of claim 17 further comprising dissociating the isolated polynucleotide molecule from the substrate prior to step (b).

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

21. The method of claim 1 wherein the random primers are about 2 kb apart from their neighboring primers.

22. 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.

23. The method of claim 1 wherein the sequence-biased amplification is a locus-biased amplification which is defined as a hybrid of generic amplification and locus-specific amplification and is performed in the presence of both random primers and locus-specific primers.

24. The method of claim 23 wherein the locus-specific primers bind to a common sequence within, or flanking, the locus shared by all or a majority of the alleles occupying the locus.

25. The method of claim 24 wherein the sequence flanking the locus is located outside the locus but the distance between the sequence flanking the locus and the nearby terminus of the locus is within 500 nucleotides.

26. The method of claim 1 wherein in the sequence-biased amplification the sequence-specific primers are end-specific primers.

27. The method of claim 26 wherein the end-specific primers anneal to the isolated polynucleotide molecule or the fragment thereof within 500 nucleotides from an end of the isolated polynucleotide molecule or the fragment thereof.

28. The method of claim 1 wherein in the sequence-specific amplification the center-specific primers are degenerate primers whose sequence has several possible bases at certain positions.

29. The method of claim 1 wherein the end-specific primers anneal to the isolated polynucleotide molecule or the fragment thereof within 500 nucleotides from an end of the isolated polynucleotide molecule or the fragment thereof.

30. 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, wherein

(i) the amplification is a sequence-biased amplification which is defined as a hybrid of generic amplification and sequence-specific amplification performed in the presence of both random primers and sequence-specific primers, or

(ii) the amplification is a sequence-specific amplification performed in the presence of center-specific primers and end-specific primers.

31. The method of claim 30 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.

32. The method of claim 31 wherein different separation groups are attached to different targeting elements.

33. The method of claim 31 wherein identical separation groups are attached to different targeting elements.

34. The method of claim 31 wherein at least three different polynucleotide molecules of interest are isolated.

35. The method of claim 30 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.

36. 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, wherein

(i) the amplification is a sequence-biased amplification which is defined as a hybrid of generic amplification and sequence-specific amplification performed in the presence of both random primers and sequence-specific primers, or

(ii) the amplification is a sequence-specific amplification performed in the presence of center-specific primers and end-specific primers.