Compositions and methods for performing a stringent wash step in hybridization applications

Patent metadata

Field	Value
Publication number	US-9388456-B2
Application number	US-200913203149-A
Country	US
Kind code	B2
Filing date	Dec 2, 2009
Priority date	Feb 26, 2009
Publication date	Jul 12, 2016
Grant date	Jul 12, 2016

Abstract

The invention provides methods and compositions for performing a stringent wash step in a hybridization assay between at least one molecule and a target. The invention may, for example, eliminate the use of, or reduce the dependence on formamide in the stringent wash step. Compositions for use in the invention include an aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature non-complementary sequences in a hybridization product.

First claim

The invention claimed is:

1. A method for performing a stringent wash step in a hybridization application comprising:
a) providing a hybridization product comprising a first nucleic acid sequence hybridized to a second nucleic acid sequence within a cell having a sample morphology,
b) providing an aqueous composition for performing a stringent wash, said composition comprising at least one polar aprotic solvent in an amount effective to denature non-complementary sequences in a hybridization product, a hybridization solution, and 5%-40% dextran sulfate, wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO) and has lactone, sulfone, sulfite, or carbonate functionality, and
c) combining the hybridization product and the aqueous composition at a temperature of 82° C. or less for at least a time period sufficient to denature any non-complementary binding between the first and second nucleic acid sequences within the cell such that the sample morphology is preserved.

2. The method according to claim 1 , wherein a sufficient amount of energy to denature any non-complementary binding between the first and second nucleic acid sequences within the cell is provided.

3. The method according to claim 2 , wherein the energy is provided by heating the combination produced in (c).

4. The method according to claim 3 , wherein the combination produced in (c) is heated to less than 70° C.

5. The method according to claim 1 , wherein the denaturation of any non-complementary binding between the first and second nucleic acid sequences within the cell occurs in less than 1 hour.

6. The method according to claim 1 , wherein the first nucleic acid sequence is in a biological sample.

7. The method according to claim 6 , wherein the biological sample is a cytology or histology sample.

8. The method according to claim 1 , wherein the aqueous composition comprises one phase at room temperature.

9. The method according to claim 1 , wherein the aqueous composition comprises multiple phases at room temperature.

10. The method according to claim 9 , wherein the aqueous composition comprises two phases at room temperature.

11. The method according to claim 9 , wherein the phases of the aqueous composition are mixed.

12. The method according to claim 1 , wherein the aqueous composition further comprises one or more salts.

13. The method according to claim 12 , wherein the salts are selected from the group consisting of sodium chloride, sodium phosphate, and magnesium phosphate.

14. The method according to claim 13 , the sodium chloride is present at a concentration of 1 mM to 1200 mM.

15. The method according to claim 13 , the sodium phosphate is present at a concentration of 10 mM to 500 mM.

Assignees

• Matthiesen Steen Hauge
• Dako Denmark As

Inventors

• Matthiesen Steen Hauge

Classifications

• C12Q2527/137

  Concentration of a component of medium · CPC title

• C12Q1/6832 Primary

  Enhancement of hybridisation reaction · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title