Methods for blocking nonspecific hybridizations of nucleic acid sequences

• US 20040009487A1
• Filed: 09/03/2002
• Published: 01/15/2004
• Est. Priority Date: 08/31/2001
• Status: Abandoned Application

• Current Assignee: CF Partners Acquisition III, LLC
• Original Assignee: CF Partners Acquisition III, LLC
• Inventors: Getts, Robert C., Kadushin, James M.
• Application Number:
  US10/234,069
• Publication Number:
  US 20040009487A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2549/126 using oligonucleotides as clamps
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

Methods are provided for blocking non-specific and specific hybridization of nucleic acid samples on a microarray. The method of the present invention comprises applying a blocking reagent to the microarray, wherein the blocking reagent comprises modified nucleotide bases, preferably LNA (Locked Nucleic Acid—modified bicyclic monomeric units with a 2′-O-4′-C methylene bridge). In further embodiments, the method includes applying a mixture including: a) a cDNA reagent obtained from mRNA of a target sample, the cDNA having a capture sequence; b) a dendrimer with a label for emitting a detectable signal and a second nucleotide sequence complementary to the capture sequence; and c) an blocking reagent containing LNA to a microarray, for producing a detectable signal from said label whereby a hybridization pattern is generated on the microarray.

Claims

1. A method comprising:

using a blocking reagent to reduce non-specific binding between a first nucleic acid sequence and a second nucleic acid sequence, wherein said blocking reagent comprises at least one modified nucleotide.

2. The method of claim 1, wherein said blocking reagent binds to said first nucleic acid sequence, and wherein said blocking reagent comprises a sequence of nucleic acids.

3. The method of claim 1, wherein said blocking reagent bound to said first nucleic acid has a higher melting temperature (Tm) than a second reagent bound to said first nucleic acid, said second reagent being the same sequence of nucleic acids but having, in place of said modified nucleotide, a standard nucleotide with the same base as said modified nucleotide.

4. The method of claim 1, wherein said modified nucleotide is LNA.

5. The method of claim 1, wherein said modified nucleotide is free of a peptide backbone.

6. The method of claim 1, wherein said blocking reagent comprises a poly-A nucleic acid sequence.

7. The method of claim 1, wherein said blocking reagent comprises a poly-T nucleic acid sequence.

8. A method comprising:

1) using a microarray wherein the microarray comprises a plurality of features, each of the features comprising a first set of molecules comprising first nucleotide sequences;

2) applying a sample to said microarray, wherein said sample comprises a second set of molecules comprising second nucleotide sequences for binding to any of said first nucleotide sequences on said microarray that are complementary;

3) using a blocking reagent to reduce non-specific binding between said first nucleotide sequences and said second nucleotide sequences, wherein said blocking reagent comprises a sequence of nucleic acids comprising at least one modified nucleotide.

9. The method of claim 8, wherein said blocking reagent has a higher melting temperature (Tm) when bound to a sequence of complementary standard nucleotide bases than the melting temperature of a reagent with the same sequence of nucleic acids but having, in place of said modified nucleotide, a standard nucleotide with the same base as said modified nucleotide.

10. The method of claim 8, wherein second set of molecules further comprise a label for producing a detectable signal.

11. The method of claim 8, wherein said second set of molecules further comprise dendrimers, said dendrimers comprising a label for producing a detectable signal.

12. A method comprising the steps of:

contacting a microarray with a mixture containing an oligonucleotide comprising at least one residue of LNA (Locked Nucleic Acid—

modified bicyclic monomeric units with a 2′

-O-4′

-C methylene bridge), said microarray comprising a plurality of features, said features comprising a first set of nucleotide sequences.

13. The method of claim 12, further comprising the step of contacting said microarray with labelled target molecules for producing a detectable signal.

14. The method of claim 12, wherein cDNA molecules are applied to said microarray, said microarray is washed to remove those of said cDNA molecules which had not hybridized to said microarray, and wherein labelled dendrimer is subsequently applied to said microarray for hybridization to said cDNA after hybridization of said cDNA to said microarray.

15. The method of claim 12, wherein said oligonucleotide comprising LNA is hybridized to said microarray, and wherein labelled target molecules are applied to said microarray, said oligonucleotide comprising LNA being hybridized to said microarray prior to application of said labelled target molecules.

16. The method of claim 12, wherein said mixture comprises target molecules, and said target molecules are hybridized to said oligonucleotide comprising LNA prior to contacting said microarray with said mixture.

17. A method comprising:

1) using a microarray wherein the microarray comprises a plurality of features, said features comprising probe nucleotide sequences;

2) applying a sample to said microarray, wherein said sample comprises target molecules for binding to any of said probe nucleotide sequences on said microarray that are complementary to said target molecules; and

,

3) using an LNA reagent as a blocking reagent to reduce non-specific binding between the target and probe molecules, wherein the LNA reagent is an oligonucleotide containing at least one residue of Locked Nucleic Acid, the Locked Nucleic Acid residues being modified bicyclic monomeric units with a 2′

-O-4′

-C methylene bridge.

18. The method of claim 17, wherein target molecules comprise a label for producing a detectable signal.

19. The method of claim 17, wherein said target molecules are cDNA molecules, and further comprising the following steps in the following order:

a) applying said cDNA molecules to said microarray;

b) washing said microarray to remove those of said cDNA molecules which have not hybridized to said microarray; and

c) applying dendrimer molecules to said microarray for hybridization to those of said cDNA molecules which have hybridized to said probe nucleotide sequences.

20. The method of claim 17, wherein said LNA reagent is hybridized to said microarray prior to application of said target molecules to said microarray.

21. The method of claim 17, wherein said target molecules are hybridized to said LNA reagent, and wherein said target molecules hybridized to said LNA reagent are subsequently applied to said microarray.

22. A method, said method comprising the steps of:

1) using a microarray comprising a plurality of features, said features comprising a first set of nucleotide sequences;

2) contacting said microarray with a mixture comprising;

a) a first component comprising a cDNA reagent obtained from mRNA of a target sample, said cDNA having a capture sequence;

b) a second component comprising a dendrimer having at least one first arm containing a label for producing a detectable signal and at least one second arm having a second nucleotide sequence complementary to said capture sequence; and

,

c) a third component comprising an synthetic DNA oligonucleotide containing residues of LNA (Locked Nucleic Acid—

modified bicyclic monomeric units with a 2′

-O-4′

-C methylene bridge) for use as a blocking reagent on said microarray.

23. The method of claim 22, further comprising the step of mixing said first component and said second component at a temperature and for a time sufficient to enable said first component to bind to the second component.

24. The method of claim 22, further comprising the step of incubating said mixture with said microarray to enable said first nucleotide sequence to bind to said first component, wherein such binding results in the feature emitting said detectable signal.

25. The method of claim 22, further comprising the step of forming the first component comprising said cDNA reagent by contacting said target sample mRNA with a quantity of an RT primer having the capture sequence, a reverse transcriptase, and nucleotide under conditions sufficient for initiating reverse transcription of said mRNA into said cDNA reagent.

26. The method of claim 25, further comprising the step of purging excess unhybridized RT primer from said first component prior to incubation of said mixture.

27. The method of claim 26, wherein said purging step further comprising the step of passing the first component through a spin column media.

28. The method of claim 22, further comprising scanning said microarray for detecting a signal from said label.

29. The method of claim 22, further comprising the step of washing said microarray to purge dendrimers unattached to said microarray after the incubation of said microarray and said mixture.

30. A method for detection and assay on a microarray, said method comprising the steps of:

1) incubating a mixture including;

a) a first component comprising a cDNA reagent obtained from mRNA of a target sample, said cDNA having a capture sequence; and

b) a second component comprising a dendrimer having at least one first arm containing a label for producing a detectable signal and at least one second arm having a second nucleotide sequence complementary to the capture sequence,

said mixture being incubated at a first temperature and for a time sufficient to induce the first component to bind to the second component and form a prehybridized cDNA-dendrimer complex;

2) contacting a microarray with said mixture, wherein said microarray comprises a plurality of features, said features comprising a first set of nucleotide sequences; and

,

3) incubating said microarray and said prehybridized cDNA-dendrimer complex at a second temperature and for a time sufficient to induce said prehybridized cDNA-dendrimer complex to bind any of said set of first nucleotide sequences that are complementary to any sequences of said cDNA reagent, wherein such binding results in said feature emitting said detectable signal such that a hybridization pattern is generated on said microarray.

31. An apparatus comprising:

a kit, said kit comprising a dendrimer and a blocking reagent, said blocking reagent comprising a modified nucleotide.

32. An apparatus as claimed in claim 31, wherein said kit further comprises an RT primer.

33. An apparatus as claimed in claim 31, wherein said kit further comprises an RNAse inhibitor.

34. An apparatus as claimed in claim 31, wherein said blocking reagent comprises LNA.

35. An apparatus as claimed in claim 31, wherein said kit is provided for use with a microarray.

36. An apparatus as claimed in claim 32, wherein said kit further comprises an RNAse inhibitor.