Hybridization promotion reagents

• US 5,589,335 A
• Filed: 08/11/1994
• Issued: 12/31/1996
• Est. Priority Date: 04/05/1989
• Status: Expired due to Term

• Current Assignee: Amoco Corporation (BP PLC)
• Original Assignee: Amoco Corporation (BP PLC)
• Inventors: Eldredge, John K., Collins, Mark L., Morrissey, David V., Kearney, Kevin R.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Houtteman, Scott W.
• Application Number:
  US08/290,001
• Time in Patent Office: 873 Days
• Field of Search: 435/6, 435/967, 935/78, 935/79
• US Class Current: 435/6.12
• 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 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: Y10S 435/967 Standards, controls, materials, e.g. validation studies, buffer systems

Abstract

Novel reagents are provided along with methods for their use in nucleic acid hybridization assays for detecting target nucleic acids in samples. The most preferred reagent is a mixture of guanidinium thiocyanate and tetramethylammonium:Trifluoracetate and possesses many properties for facilitating hybridization between target nucleic acid and nucleic acid probes capable of binding thereto and surprisingly results in the phenomenon of superstoichiometric labeling.

Claims

1. A reagent for performing rRNA hybridization reactions comprising a soluble salt of guanidine in a concentration of 0.7-1.7M, a soluble salt of thiocyanate in concentration of 0.7-2.4M, a soluble salt of tetramethylammonium in a concentration of 1.2-2.2M, and a soluble salt of trifluoroacetate in a concentration of 0.8-2.0M;

said reagent capable of reducing ribonuclease activity, promoting the speed of hybridization processes, providing superstoichiometric labeling and equalizing the relative strength of GC and AT base pairs.

2. The reagent of claim 1 wherein at least one soluble salt is guanidine thiocyanate.

3. The reagent of claim 1 wherein at least one said soluble salt is tetramethylammonium trifluoroacetate.

4. The reagent of claim 1 wherein said soluble salts comprise guanidine thiocyanate in a concentration of 1.2-1.7M and tetramethylammonium trifluoroacetate in a concentration of 1.2-1.6M.

5. The reagent of claim 4 wherein said guanidine thiocyanate has a concentration of 1.6M.

6. The reagent of claim 4 wherein said tetramethylammonium trifluoroacetate has a concentration of 1.25M.

7. The reagent of claim 1, wherein said rRNA is 16S rRNA.

8. The reagent of claim 2, wherein said rRNA is 16S rRNA.

9. The reagent of claim 3, wherein said rRNA is 16S rRNA.

10. The reagent of claim 4, wherein said rRNA is 16S rRNA.

11. The reagent of claim 5, wherein said rRNA is 16S rRNA.

12. The reagent of claim 6, wherein said rRNA is 16S rRNA.