# US 11,078,534 B2 · Photocleavable nucleotide reagents with high stability

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- Number: US 11,078,534 B2
- Document ID: US11078534B2
- Status: Granted
- Kind: B2
- Application number: 16/199,757
- Filed: 2018-11-26
- Published: 2019-05-30
- Granted: 2021-08-03
- Earliest claimed: 2017-11-27
- Assignees: Roche Molecular Systems, Inc.
- Applicants: Roche Molecular Systems, Inc.
- Inventors: Alexander Nierth; Hannes Kuchelmeister; Concordio Anacleto; Amar Gupta; Dieter Heindl; Igor Kozlov; Christian Wellner; Stephen Gordon Will
- Web page: https://patentgrep.com/patent/US11078534B2

## CPC

- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 21/02: with ribosyl as saccharide radical
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/686: Polymerase chain reaction [PCR]
- 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
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)

## Abstract

The present invention provides for stable nucleotide reagents used for nucleic acid amplification by PCR and RT-PCR (Reverse Transcriptase-PCR) that comprises modified nucleoside triphosphates. The present invention also provides for methods for using the modified nucleoside triphosphates for detecting the presence or absence of a target nucleic acid sequence in a sample in an amplification reaction.

## Claims

### Claim 1

A method of detecting the presence or absence of a target nucleic acid sequence in a sample comprising:
a) performing an amplifying step comprising contacting the sample with amplification reagents to produce an amplification product if the target nucleic acid sequence is present in the sample, and
b) detecting the amplification product,
wherein the amplification reagents comprise a modified nucleoside triphosphate having a photocleavable moiety and having a structure of:
where Base is a purine or pyrimidine base or an analog; and
wherein prior to step a), the modified nucleoside triphosphate is irradiated with light having a wavelength capable of cleaving the photocleavable moiety.

### Claim 2

A method of amplifying a target nucleic acid sequence in a sample comprising:
providing amplification reagents to perform an amplifying step, wherein the amplification reagents comprise a modified nucleoside triphosphate having a photocleavable moiety and having a structure of:
where Base is a purine or pyrimidine base or an analog; and
wherein prior to the amplifying step, the modified nucleoside triphosphate is irradiated with light having a wavelength capable of cleaving the photocleavable moiety.

### Claim 3

A method of reducing or preventing non-specific amplification of nucleic acid in a sample during amplification by polymerase chain reaction (PCR) comprising:
a) providing amplification reagents that are used to amplify a target nucleic acid, wherein the amplification reagents comprise a nucleic acid polymerase and a modified nucleoside triphosphate having a photocleavable moiety and having a structure of:
where Base is a purine or pyrimidine base or an analog;
b) contacting the sample with the amplification reagents at room temperature, whereby the photocleavable moiety on the modified nucleoside triphosphate prevents processing by the nucleic acid polymerase, thereby suppressing formation of non-specific amplification products from room temperature activity of the nucleic acid polymerase;
c) prior to amplifying, irradiating the modified nucleoside triphosphate with light having a wavelength capable of cleaving the photocleavable moiety to release free nucleoside triphosphate; and
d) amplifying the target nucleic acid by PCR.

### Claim 4

A modified nucleoside triphosphate having a photocleavable moiety and having a structure of:
where Base is a purine or pyrimidine base or an analog.

### Claim 5 (depends on 4)

The modified nucleoside triphosphate of claim 4 characterized by having greater thermostability compared to an unmodified nucleoside triphosphate.

### Claim 6 (depends on 4)

A kit comprising the modified nucleoside triphosphate of claim 4 and further comprising reagents necessary for performing an amplification reaction.

### Claim 7 (depends on 5)

A kit comprising the modified nucleoside triphosphate of claim 5 and further comprising reagents necessary for performing an amplification reaction.