Method for isolating and recovering target DNA or RNA molecules having a desired nucleotide sequence

• US 6,268,133 B1
• Filed: 06/24/1998
• Issued: 07/31/2001
• Est. Priority Date: 06/25/1997
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Li, Wu-bo, Jesse, Joel, Nisson, Paul E.
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US09/103,577
• Time in Patent Office: 1,133 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

The invention generally concerns the use of amino acid denaturants for denaturing or separating double stranded nucleic acid molecules. More specifically, the present invention provides a method for the rapid isolation and recovery of a desired target DNA or RNA molecules from a mixture or library containing such molecules. The method involves the use of haptenylated probes and amino acid denaturants to select the desired molecules and eliminate the undesired library members from a sample. The invention also provides a method in which larger or full-length nucleic acid molecules can be isolated from the subpopulation of desired molecules.

Claims

1. A method for denaturing or separating double-stranded nucleic acid molecules, said method comprising contacting one or more double-stranded nucleic acid molecules with a denaturant selected from the group consisting of

(a) one or more amino acids comprising:

glycine, α

-alanine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine,

(b) imidazole, and

(c) one or more amino acid denaturants plus imidazole,

thereby forming single-stranded nucleic acid molecules.

2. The method of claim 1, wherein said amino acid denaturants are selected from the group consisting of one or more amino acids, polyamino acids, and combinations thereof, wherein said amino acids denaturants denature or separate double-stranded nucleic acid molecules.

3. The method of claim 2, wherein said amino acid denaturants are natural or unnatural amino acids.

4. The method of claim 3, wherein said polyamino acids comprise two or more amino acids.

5. The method of claim 3, wherein said amino acid denaturants are selected from the group consisting of glycine, α

-alanine, arginine, asparagine, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

6. The method of claim 1, wherein said denaturant is glycine.

7. The method of claim 1, wherein the concentration of said denaturants ranges from about 1 mM to about 500 mM.

8. The method of claim 7, wherein said concentration ranges from about 5 mM to about 50 mM.

9. The method of claim 8, wherein said concentration is about 10 mM.

10. A method of recovering one or more desired target nucleic acid molecules from a population of nucleic acid molecules comprising:

(a) contacting said population with one or more haptenylated probes to permit said probe to hybridize to said desired target molecules thereby forming one or more hybridized molecules; and

(b) isolating said desired target nucleic acid molecules from said probes by contacting said hybridized molecules with a denaturant selected from the group consisting of

(i) one or more amino acids comprising;

glycine, a-alanine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine,

(ii) imidazole, and

(iii) one or more amino acid denaturants plus imidazole.

11. The method of claim 10, wherein said population of nucleic acid molecules is single-stranded DNA.

12. The method of claim 11, wherein said single-stranded DNA is circular.

13. The method of claim 11, wherein said single-stranded DNA is linear.

14. The method of claim 11, wherein said single-stranded DNA is selected from the group consisting of single-stranded plasmids, single-stranded cosmids, and single-stranded phagemids.

15. The method of claim 10, wherein said population of nucleic acid molecules is a cDNA library.

16. The method of claim 10, wherein said haptenylated probes are bound to a support.

17. The method of claim 16, wherein said support comprises one or more binding ligands.

18. The method of claim 17, wherein said probes are bound to said support by a ligand-hapten interaction.

19. The method of claim 10, wherein said population of nucleic acid molecules is double-stranded DNA.

20. The method of claim 19, further comprising treating said double-stranded nucleic acid molecules to render such molecules single-stranded.

21. The method of claim 20, wherein said treatment comprises contacting said double-stranded nucleic acid molecule with a denaturant selected from the group consisting of one or more amino acid denaturants, imidazole, and one or more amino acid denaturants plus imidazole.

22. The method of claim 20, wherein said treatment comprises degradation of one strand of said double-stranded nucleic acid molecules.

23. The method of claim 22, wherein said degradation comprises the use of Gene II protein and Exonuclease III.

24. The method of claim 10, further comprising (c) synthesizing a nucleic acid molecule complementary to said desired target molecules and forming double-stranded nucleic acid molecules.

25. The method of claim 24, wherein said conditions comprise the use of one or more primer nucleic acid molecules and one or more nucleotides.

26. The method of claim 25, wherein said nucleotides confer nuclease resistance to said synthesized nucleic acid molecule.

27. The method of claim 26, wherein said nucleotides are nucleotide analogs.

28. The method of claim 27, wherein said nucleotide analogs are a methylated nucleotides.

29. The method of claim 28, wherein said methylated nucleotides are 5-methyldeoxycytosine.

30. The method of claim 26, further comprising digesting said double-stranded nucleic acid molecules with one or more nuclease.

31. The method of claim 30, further comprising transforming said digested molecules into one or more host cells.

32. The method of claim 24, further comprising transforming said double-stranded molecules into one or more host cells.

33. The method of claim 10, wherein said probes are degenerate probes.

34. The method of claim 33, wherein said degenerate probes comprise one or more universal nucleotides.

35. The method of claim 34, wherein said degenerate probes comprise one or more nucleotides selected from the group consisting of dP and dK.

36. The method of claim 10, further comprising enriching for larger or full-length desired nucleic acid molecules.

37. The method of claim 36, wherein said enrichment comprises separating the desired nucleic acid molecules according to size.

38. The method of claim 37, wherein said method comprises amplifying the desired nucleic acid molecules prior to size separation.

39. The method of claim 10, wherein said probes comprise a Kozac sequence.

40. The method of claim 39, wherein said probes are degenerate probes.

41. The method of claim 33, wherein said degenerate probes are to a Kozac sequence.