# US 9,434,988 B2 · RNase H-based assays utilizing modified RNA monomers

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- Number: US 9,434,988 B2
- Document ID: US9434988B2
- Status: Granted
- Kind: B2
- Application number: 13/429,077
- Filed: 2012-03-23
- Published: 2012-10-11
- Granted: 2016-09-06
- Earliest claimed: 2008-04-30
- Assignees: Integrated Dna Technologies, Inc.
- Applicants: Mark Aaron Behlke; Scott D. Rose; Joseph Dobosy; Joseph Alan Walder
- Inventors: Mark Aaron Behlke; Scott D. Rose; Joseph Dobosy; Joseph Alan Walder
- Web page: https://patentgrep.com/patent/US9434988B2

## CPC

- C12Q 1/6858: Allele-specific amplification
- 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

The present invention provides methods of cleaving a nucleic acid strand to initiate, assist, monitor or perform biological assays.

## Claims

### Claim 1

A method of amplifying a target DNA sequence, the method comprising the steps of:
(a) providing a reaction mixture at a starting temperature at or below 40° C. wherein the reaction mixture comprises:
(i) a first oligonucleotide primer having a cleavage domain positioned 5′ of a blocking group, the blocking group linked at or near the end of the 3′-end of the oligonucleotide primer, wherein the blocking group prevents primer extension and/or PCR,
(ii) a sample nucleic acid that may or may not have the target sequence,
(iii) a cleaving enzyme, wherein the cleaving enzyme is an RNase H2 enzyme,
(iv) a polymerase, and
(v) optionally, a second oligonucleotide primer in reverse orientation to support PCR;
(b) elevating the temperature of the reaction mixture to at or above 50° C. to increase the activity of the RNase H2 enzyme;
(c) hybridizing the blocked oligonucleotide primer to the target DNA sequence to form a double-stranded substrate;
(d) cleaving the hybridized oligonucleotide primer with the cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the oligonucleotide primer; and
(e) extending the oligonucleotide primer with the polymerase,
wherein the RNase H2 enzyme has, at the starting temperature, less than about 16% of the activity that the RNase H2 enzyme has at the elevated temperature in step (b).

### Claim 2 (depends on 1)

The method of claim 1 wherein the cleavage domain is a single RNA residue.

### Claim 3 (depends on 1)

The method of claim 1 wherein the amplification is performed in a PCR assay that is used to discriminate between variant alleles.

### Claim 4 (depends on 3)

The method of claim 3 wherein the PCR assay is used to quantitate the abundance of the target nucleic acid sequence in the sample.

### Claim 5 (depends on 1)

The method of claim 1, wherein the starting temperature is at or below 37° C.

### Claim 6 (depends on 1)

The method of claim 1 wherein the RNase H2 enzyme has, at the starting temperature, less than about 15% of the activity that the RNase H2 enzyme has at the elevated temperature in step (b).

### Claim 7 (depends on 1)

The method of claim 1 wherein the RNase H2 enzyme has, at the starting temperature, less than about 5% of the activity that the RNase H2 enzyme has at the elevated temperature in step (b).

### Claim 8 (depends on 1)

The method of claim 1 wherein the RNase H2 enzyme has, at the starting temperature, less than about 1% of the activity that the RNase H2 enzyme has at the elevated temperature in step (b).

### Claim 9 (depends on 1)

The method of claim 1 wherein the RNase H2 enzyme is Pyrococcus abyssi RNase H2.

### Claim 10 (depends on 1)

The method of claim 1, wherein the RNase H2 enzyme is encoded by SEQ ID NO: 4.

### Claim 11 (depends on 1)

The method of claim 1, wherein the RNase H2 enzyme is reversibly inactivated either by chemical modification or by a blocking antibody.