Methods and systems for detection and isolation of a nucleotide sequence

• US 20080096191A1
• Filed: 08/14/2007
• Published: 04/24/2008
• Est. Priority Date: 06/24/2002
• Status: Abandoned Application

• Current Assignee: Exiqon A/S (Qiagen NV)
• Original Assignee: Exiqon A/S (Qiagen NV)
• Inventors: Kauppinen, Sakari, Jacobsen, Nana
• Application Number:
  US11/893,108
• Publication Number:
  US 20080096191A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

A method for isolating nucleic acid molecules having a repeating nucleotide sequence or a homopolymeric nucleotide sequence, e.g. a poly A stretch, is described. In particular, the method uses oligomeric capture probes spiked with various amounts of locked nucleic acid (LNA). The invention further describes methods for the isolation of RNA molecules, for example polyadenylated mRNA molecules, which overcome the problems of rapid RNA degradation during isolation and analysis of such nucleic acid molecules. This is of major clinical and diagnostic importance, especially when dealing with RNA viruses, such as retroviruses or when analyzing rare or low-abundant mRNAs or mRNAs from biopsies or tissues enriched with RNases.

Claims

1. A method for detecting and/or isolating a target nucleic acid molecule having a homopolymeric sequence comprising:

a) treating a sample containing nucleic acid molecules with a lysis buffer comprising a chaotropic agent;

b) treating the sample under high stringency hybridization conditions using low salt concentration with an LNA oligonucleotide covalently attached to a solid support and comprising at least twenty repeating consecutive nucleotides;

c) performing a washing step to remove excess material,

wherein said homopolymeric sequence is a poly(A) tail of a eukaryotic mRNA.

6. The method of claim 1, wherein the LNA oligonucleotide capture probe is synthesized with an anthraquinone moiety and a linker at the 5′

-end or the 3′

-end of said oligonucleotide.

7. The method of claim 6 wherein said linker is selected from the group consisting of one or more of a hexaethylene glycol monomer, dimer, trimer, tetramer, pentamer, hexamer, or higher hexaethylene glycol polymer;

a poly-T sequence of 10-50 nucleotides in length;

a poly-C sequence of 10-50 nucleotides in length or longer; and

a non-base sequence of 10-50 nucleotide units in length or longer.

8. The method of claim 1 wherein said solid support is a polymer support selected from the group consisting of a microtiter plate, polystyrene beads, latex beads, a polymer microscope slide or a polymer-coated microscope slide and a microfluidic slide.

9. The method of claim 1 wherein the LNA oligonucleotide is complementary to a homopolymeric nucleotide comprising at least about one nucleobase that is different than the bases comprising the homopolymeric nucleic acid sequence.

13. The method of claim 1, wherein the LNA oligonucleotide comprises at least about thirty repeating consecutive nucleotides.

14. The method of claim 1, wherein the LNA oligonucleotide comprises at least about forty repeating consecutive nucleotides.

15. The method of claim 1, wherein the LNA oligonucleotide comprises at least about fifty repeating consecutive nucleotides.

17. The method of claim 7, a covalent coupling onto a solid polymer support of said LNA oligonucleotide is carried out via excitation of the anthraquinone moiety using UV light.

22. The method of claim 1, wherein the LNA oligonucleotide is selected from the following table:

24. The method of claim 22, wherein the LNA oligonucleotide is selected from the group of oligonucleotides corresponding to Compounds 2 to 10 herein having an anthraquinone in the 5′

position instead of biotin.

25. The method of claim 1, wherein the LNA oligonucleotide is selected from the group consisting of a oligonucleotides corresponding to Compounds 2 to 18 herein having an anthraquinone in the 5′

position and a linker which is selected from the group consisting of one or more of a hexaethylene glycol monomer, dimer, trimer, tetramer, pentamer, hexamer, or higher hexaethylene glycol polymer;

a poly-T sequence of 10-50 nucleotides in length and a poly-C sequence of 10-50 nucleotides in length or longer.

26. The method of claim 1, wherein the LNA oligonucleotide molecule is selected from the group consisting of oligonucleotides corresponding to Compounds 2 to 10 herein without the biotin substitution in the 5′

position.

29. The method of claim 1, wherein the LNA oligonucleotide comprises at least one nucleotide having a nucleobase that is different from the nucleobases of the remaining oligonucleotide sequence.

30. The method of claim 1, wherein the −

1 residue of the LNA oligonucleotide molecule 3′ and

/or 5′

end is an LNA residue.

31. The method of claim 1, wherein the LNA oligonucleotide comprises at least about one or more alpha-L LNA monomers.

32. The method of claim 1, wherein the LNA oligonucleotide comprises at least about one or more xylo-LNA monomers.

33. The method of claim 1, wherein the LNA oligonucleotide comprises at least about 20 to 50 percent LNA residues based on total residues of the LNA oligonucleotide.

34. The method of claim 1, wherein the LNA oligonucleotide comprises at least about two or more consecutive LNA molecules.

35. The method of claim 1, wherein the LNA oligonucleotide comprises modified and non-modified nucleotide molecules.

36. The method of claim 1, wherein the LNA oligonucleotide comprises a compound of the formula:

5′

-Y^q—

(X^p—

Yⁿ)m-X^p-Z-3′

wherein X is an LNA monomer, Y is a DNA monomer;

Z represents an optional DNA monomer;

p is an integer from about 1 to about 15;

n is an integer from about 1 to about 15 or n represents 0;

q is an integer from about 1 to about 10 or q=0; and

m is an integer from about 5 to about 20.

39. The method of claim 1, wherein the LNA oligonucleotide is complementary to the sequence it is designed to detect and/or isolate.

40. The method of claim 39 wherein the LNA oligonucleotide has at least one base pair difference to a complementary sequence it is designed to detect and/or isolate.

41. The method according to claim 40 wherein the LNA oligonucleotide can detect at least about one base pair difference between a complementary poly-repetitive base sequence and the LNA/DNA oligonucleotide.

42. The method of claim 1, wherein the LNA oligonucleotide comprises a fluorophore moiety and a quencher moiety, positioned in such a way that a hybridized state of the oligonucleotide can be distinguished from an unbound state of the oligonucleotide by an increase in the fluorescent signal from the nucleotide.

43. The method of claim 1, wherein the Tm of the LNA oligonucleotide is between about 50°

C. to about 70°

C. when the LNA oligonucleotide hybridizes to its complementary sequence.

44. The method of claim 1, wherein the chaotropic agent is guanidinium thiocyanate.

45. The method of claim 44 wherein the guanidinium thiocyanate is at a concentration of at least about 2M.

46. The method of claim 44 wherein the concentration of the guanidinium thiocyanate is at a concentration of at least about 3M.

47. The method of claim 44 wherein the concentration of the guanidinium thiocyanate is at a concentration of at least about 4M.

48. The method of claim 44 wherein the LNA oligonucleotide hybridizes to the target nucleic acid molecule at a temperature in the range of 20-65°

C.

53. The method of claim 1, wherein the LNA oligonucleotide is adapted for use as a TaqMan probe or Molecular Beacon.

56. The method of claim 1, wherein the eukaryotic mRNA is isolated using the covalently coupled LNA oligonucleotide, and detected with nucleic acid probes, using

(i) chemiluminescence,

(ii) bioluminescence,

(iii) ligands incorporated into the nucleic acid probes, or

(iv) biotin-labeled nucleic acid probes.

57. The method of claim 56, wherein the eukaryotic mRNA is detected using a nucleic acid probe comprising LNA combined with a tyramide signal amplification system.

58. The method of claim 56, wherein the eukaryotic mRNA is detected using a nucleic acid probe comprising LNA, containing a complementary overhang to a free arm in a dendrimer or a branched oligonucleotide conjugated with several digoxigenin, fluorescein isothiocyanate or biotin molecules or fluorochrome molecules, combined with alkaline phosphatase-conjugated or horse radish peroxidase-conjugated anti-digoxigenin, anti-fluorescein isothiocyanate antibodies or streptavidin or detection of fluorescence from the excited fluorochromes.

59. The method of claim 1, further comprising contacting the sample with a polymerase and at least one nucleotide.

60. The method of claim 59, further comprising performing said contacting under conditions suitable for generating a plurality of copies of said eukaryotic mRNA.

77. The method of claim 59, further comprising adding a DNA polymerase, RNaseH and E. coli DNA ligase after conversion of the eukaryotic polyadenylated mRNA to first strand complementary DNA under conditions suitable for generating double stranded complementary DNA

79. The method of claim 77 where the LNA oligonucleotide complementary to the poly(A) tail sequence in eukaryotic mRNA contains an anchor sequence for a RNA polymerase, such as T7 RNA polymerase.

116. The method of claim 1, wherein the genomic RNA is isolated from retroviruses.

117. The method of claim 116, wherein the retrovirus is HIV.

128. The method of claim 1 wherein said chaotropic agent is GuSCN in a concentration of at least 4 M.

129. The method of claim 1 wherein the method further comprises the step of binding the LNA oligonucleotide to nucleic acids from the sample in a binding buffer containing NaCl or LiCl.

130. The method of claim 129 where NaCl or LiCl is at a concentration less than 100 mM.

131. The method of claim 129 where NaCl or the LiCl is at a concentration less than 50 mM.

132. The method of claim 129 wherein NaCl or LiCl is at a concentration less than 25 mM.

133. The method of claim 1 wherein detection or hybridisation is carried out at least 25°

C.

134. The method of claim 1 wherein detection or hybridisation is carried out at least 37°

C.

135. The method of claim 1 wherein detection or hybridisation is carried out at least 50°

C.

136. The method according to claim 56 comprising detecting chemiluminescence using enzyme-conjugated nucleic acid probes.

137. The method according to claim 56 comprising detecting bioluminescence using firefly or bacterial luciferase or green fluorescent protein as reporter molecule.

138. The method according to claim 56 comprising detecting bioluminescence using firefly or bacterial luciferase or green fluorescent protein as reporter molecule.

139. The method according to claim 56 comprising detecting digoxigenin (DIG), fluorescein isothiocyanate (FITC), or biotin incorporated into the nucleic acid probes.

140. The method of claim 79 wherein the RNA polymerase comprises a T7 RNA polymerase.

2-5. -5. (canceled)

10-12. -12. (canceled)

16. (canceled)

18-21. -21. (canceled)

23. (canceled)

27-28. -28. (canceled)

37-38. -38. (canceled)

49-52. -52. (canceled)

54-55. -55. (canceled)

61-76. -76. (canceled)

78. (canceled)

80. The method of claim 78 further comprising adding an RNA polymerase, such as T7 RNA polymerase, under conditions suitable for generating a plurality of RNA copies of said nucleic acid molecule.

81-115. -115. (canceled)

118-127. -127. (canceled)