# US 5,702,896 A · Methods for improving the sensitivity of hybridization assays

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- Number: US 5,702,896 A
- Document ID: US5702896A
- Status: Granted
- Kind: A
- Application number: 08/598,142
- Filed: 1996-02-07
- Granted: 1997-12-30
- Earliest claimed: 1991-02-27
- Assignees: Amoco Corporation
- Inventors: Mark L. Collins; Cecile Blomquist; Massimo Lombardo; John Eldredge
- Web page: https://patentgrep.com/patent/US5702896A

## CPC

- C12Q 1/6813: Hybridisation assays
- C12Q 1/6832: Enhancement of hybridisation reaction
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Methods for improving the sensitivity of hybridization assays which reduce non-specific binding (NSB) and non-specific hybridization (NSH) are disclosed. The methods include a washing method utilizing tetraalkylammonium salts at high temperatures, and release methods in which a probe-target complex is released from a solid support and recaptured. Use of both the washing and release methods results in substantial reduction in NSB and NSH without performing several rounds of release and recapture of the target nucleic acids.

## Claims

### Claim 1

A method for increasing the signal to noise ratio in a capture/detection hybridization assay, comprising:
a) providing a quaternary complex comprising:
i) a target nucleic acid having a first and a second region;
ii) a capture probe comprising a first region which is a homopolynucleotide (dA) tail, and a second region which is perfectly complementary to the first region of the target nucleic acid, the second region having a length of from 24 to 40 nucleotides;
iii) a detection probe which is complementary to the second region of the target nucleic acid; and
iv) a homooligonucleotide of (dT) bound to a solid support, the homooligonucleotide of (dT) having a length of from 10 to 14 nucleotides; and
b) washing the complex of step a) with a solution comprising a tetramethylammonium salt, having a molarity of about 3.0, at a temperature of from 60.degree. C. to 70.degree. C. to elute non-specifically bound detector probe.

### Claim 2

A method for increasing the signal to noise ratio in a capture/detection hybridization assay, comprising:
a) providing a quaternary complex comprising:
i) a target nucleic acid having a first and a second region;
ii) a capture probe comprising a first region which is a homopolynucleotide (dT) tail, and a second region which is perfectly complementary to the first region of the target nucleic acid, the second region having a length of from 24 to 40 nucleotides;
iii) a detection probe which is complementary to the second region of the target nucleic acid; and
iv) a homooligonucleotide of (dA) bound to a solid support, the homooligonucleotide of (dA) having a length of from 10 to 14 nucleotides; and
b) washing the complex of step a) with a solution comprising a tetramethylammonium salt, having a molarity of about 3.0, at a temperature of from 60.degree. C. to 70.degree. C. to elute non-specifically bound detector probe.