# US 7,745,125 B2 · 2′-terminator related pyrophosphorolysis activated polymerization

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- Number: US 7,745,125 B2
- Document ID: US7745125B2
- Status: Granted
- Kind: B2
- Application number: 11/583,606
- Filed: 2006-10-18
- Published: 2007-07-05
- Granted: 2010-06-29
- Earliest claimed: 2004-06-28
- Assignees: Roche Molecular Systems, Inc.
- Inventors: David H. Gelfand; Keith A. Bauer; Amar P. Gupta; Veeraiah Bodepudi; John Niemiec
- Web page: https://patentgrep.com/patent/US7745125B2

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/156: Polymorphic or mutational markers
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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 reaction mixtures that include blocked oligonucleotides comprising 2′-terminator nucleotides. The blocked oligonucleotides are rendered extendible when the 2′-terminator nucleotides are removed from the oligonucleotides, e.g., via pyrophosphorolysis. The reaction mixtures can be used in various nucleic acid polymerization and/or amplification assays, among many other applications. In addition to reaction mixtures, the invention also provides related methods and reaction mixtures.

## Claims

### Claim 1

A method of removing a nucleotide from an oligonucleotide, the method comprising:
incubating at least one target nucleic acid with:
pyrophosphate;
at least a first biocatalyst comprising a pyrophosphorolysis activity, and
at least one oligonucleotide comprising a 2′-terminator nucleotide, which oligonucleotide is at least partially complementary to at least a first subsequence of the target nucleic acid,
under conditions whereby the first biocatalyst removes at least the 2′-terminator nucleotide from the oligonucleotide to produce a removed 2′-terminator nucleotide and a shortened oligonucleotide, thereby removing the nucleotide from the oligonucleotide.

### Claim 2 (depends on 1)

The method of claim 1, wherein the oligonucleotide comprises the 2′-terminator nucleotide at a 3′-terminus.

### Claim 3 (depends on 1)

The method of claim 1, wherein the oligonucleotide comprises a primer nucleic acid or a probe nucleic acid.

### Claim 4 (depends on 1)

The method of claim 1, wherein the 2′-terminator nucleotide is non-extendible by one or more nucleotide incorporating biocatalysts selected from the group consisting of: a G46E E678G CS5 DNA polymerase, a G46E L329A E678G CS5 DNA polymerase, a G46E L329A D640G S671F CS5 DNA polymerase, a G46E L329A D640G S671F E678G CS5 DNA polymerase, a G46E E678G CS6 DNA polymerase, a ΔZO5R polymerase, a E615G Taq DNA polymerase, a Thermus flavus polymerase, a TMA-25 polymerase, a E678G TMA-25 polymerase, a TMA-30 polymerase, a E678G TMA-30 polymerase, a Tth DNA polymerase, a Thermus species SPS-17 polymerase, a E615G Taq polymerase, a Thermus ZO5R polymerase, a T7 DNA polymerase, a Kornberg DNA polymerase I, a Klenow DNA polymerase, a Taq DNA polymerase, a Micrococcal DNA polymerase, an alpha DNA polymerase, a reverse transcriptase, an AMV reverse transcriptase, an M-MuLV reverse transcriptase, a DNA polymerase, a T4 DNA polymerase, a ribonucleotide analog-incorporating DNA polymerase, and a ribonucleotide incorporating DNA polymerase.

### Claim 5 (depends on 1)

The method of claim 1, wherein the target nucleic acid comprises at least one polymorphic nucleotide position, and wherein the method comprises detecting removal of the 2′-terminator nucleotide from the oligonucleotide, which removal correlates with the oligonucleotide comprising at least one nucleotide position that corresponds to the polymorphic nucleotide position.

### Claim 6 (depends on 5)

The method of claim 5, wherein the 2′-terminator nucleotide corresponds to the polymorphic nucleotide position.

### Claim 7 (depends on 1)

The method of claim 1, wherein the oligonucleotide comprises at least one label, and wherein the method comprises detecting a detectable signal emitted from the label.

### Claim 8 (depends on 7)

The method of claim 7, wherein the label comprises a donor moiety and/or an acceptor moiety and the detectable signal comprises light emission, and wherein the method comprises incubating the target nucleic acid with the first biocatalyst, the oligonucleotide, and at least one soluble light emission modifier and detecting the light emission from the donor moiety and/or the acceptor moiety.

### Claim 9 (depends on 8)

The method of claim 8, wherein the 2′-terminator nucleotide comprises the donor moiety and/or the acceptor moiety.

### Claim 10 (depends on 1)

The method of claim 1, wherein the first biocatalyst comprises a nucleotide incorporating activity, and wherein the method comprises incubating the target nucleic acid with the first biocatalyst, the shortened oligonucleotide, and at least one additional nucleotide under conditions whereby the first biocatalyst incorporates the additional nucleotide at a terminus of the shortened oligonucleotide to produce an extended oligonucleotide; or,
wherein the method comprises incubating the target nucleic acid with at least a second biocatalyst comprising a nucleotide incorporating activity, the shortened oligonucleotide, and at least one additional nucleotide under conditions whereby the second biocatalyst incorporates the additional nucleotide at a terminus of the shortened oligonucleotide to produce an extended oligonucleotide.

### Claim 11 (depends on 10)

The method of claim 10, wherein the first and/or second biocatalyst comprises an enzyme selected from the group consisting of: a polymerase and a reverse transcriptase.

### Claim 12 (depends on 10)

The method of claim 10, wherein the nucleotide incorporating activity comprises a polymerase activity and/or a ligase activity.

### Claim 13 (depends on 10)

The method of claim 10, wherein one or more nucleotides of the oligonucleotide extend beyond a terminus of the target nucleic acid when the oligonucleotide and the target nucleic acid hybridize to form a hybridized nucleic acid.

### Claim 14 (depends on 10)

The method of claim 10, wherein at least one additional oligonucleotide comprises the additional nucleotide.

### Claim 15 (depends on 10)

The method of claim 10, wherein the additional nucleotide comprises an extendible nucleotide.

### Claim 16 (depends on 10)

The method of claim 10, wherein the first and/or second biocatalyst comprises a CS5 DNA polymerase comprising one or more mutations at amino acid positions selected from the group consisting of: G46, L329, Q601, D640, I669, S671, and E678.

### Claim 17 (depends on 16)

The method of claim 16, wherein the mutations comprise a G46E mutation, an L329A mutation, a Q601R mutation, a D640G mutation, an I669F mutation, an S671F mutation, and/or an E678G mutation.

### Claim 18 (depends on 10)

The method of claim 10, wherein the additional nucleotide comprises at least one label, and wherein the method comprises detecting a detectable signal emitted from the label.

### Claim 19 (depends on 18)

The method of claim 18, wherein the label comprises a donor moiety and/or an acceptor moiety and the detectable signal comprises light emission, and wherein the method comprises incubating the target nucleic acid with at least one soluble light emission modifier and detecting the light emission from the label.

### Claim 20 (depends on 10)

The method of claim 10, comprising incubating the target nucleic acid with at least one probe nucleic acid that comprises at least one label, which probe nucleic acid is at least partially complementary to at least a second subsequence of the target nucleic acid, and detecting a detectable signal emitted from the label of the probe nucleic acid or a fragment thereof.

### Claim 21 (depends on 20)

The method of claim 20, wherein the detectable signal comprises light emission, and wherein the method further comprises incubating the target nucleic acid with at least one soluble light emission modifier and detecting the light emission from the label.

### Claim 22 (depends on 20)

The method of claim 20, wherein the probe nucleic acid comprises a 5′-nuclease probe and the first and/or second biocatalyst extends the shortened oligonucleotide in a 5′ to 3′ direction and comprises a 5′ to 3′ exonuclease activity.

### Claim 23 (depends on 20)

The method of claim 20, wherein the probe nucleic acid comprises a hybridization probe and/or a hairpin probe.

### Claim 24 (depends on 10)

The method of claim 10, wherein the target nucleic acid comprises at least one polymorphic nucleotide position, and wherein the method comprises detecting extension of the shortened oligonucleotide, which extension correlates with the extended oligonucleotide comprising at least one nucleotide position that corresponds to the polymorphic nucleotide position.

### Claim 25 (depends on 24)

The method of claim 24, wherein the 2′-terminator nucleotide corresponds to the polymorphic nucleotide position.

### Claim 26 (depends on 1)

The method of claim 1, wherein the 2′-terminator nucleotide comprises the formula
wherein
R1 is H, OH, a hydrophilic group, or a hydrophobic group;
B is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or combinations thereof;
BG is a blocking group;
Z is O or CH2; and represents a single or double bond; and
wherein BG is selected from the group consisting of: CN, NO2, N3, Cl, Br, I, a phosphate group, an aldehyde group, an ester group, an amino group, and combinations thereof.

### Claim 27 (depends on 1)

The method of claim 1, wherein the 2′-terminator nucleotide comprises the formula
wherein
R1 is H or OH;
B is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or combinations thereof;
BG is a blocking group;
Z is O or CH2; and represents a single or double bond; and
wherein BG is selected from the group consisting of: CN, NO2, a phosphate group, an aldehyde group, an amino group, and combinations thereof.

### Claim 28 (depends on 1)

The method of claim 1, wherein the 2′-terminator nucleotide comprises the formula
wherein
R1 is OH;
B is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or combinations thereof;
BG is a blocking group;
Z is O or CH2; and represents a single or double bond; and
wherein BG is selected from the group consisting of: CN, NO2, a phosphate group, an aldehyde group, an amino group, and combinations thereof.

### Claim 29 (depends on 1)

The method of claim 1, wherein the 2′-terminator nucleotide comprises the formula
wherein
R1 is H, OH, a hydrophilic group, or a hydrophobic group;
B is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or combinations thereof;
BG is a blocking group;
Z is O or CH2; and represents a single or double bond; and
wherein BG comprises a phosphate group comprising the formula:
wherein
Q is O, S, or NH;
X is H, OH, CH3, BH3, F, or SeH; and,
Z is O, S, or Se; or,
wherein
Q is O, S, or NH;
X is O, S, or NH;
Z is O, S, or Se; and,
R is an alkyl group, an alkenyl group, or an alkynyl group; or,
wherein
Q is O, S, or NH;
X is O, S, or NH;
Z is O, S, or Se;
L is —CONH(CH2)nNH—, —CO(CH2)nNH—, or —CONH(CH2CH2O)nCH2CH2NH—;
n is an integer greater than 0; and,
R is NH2, SH, COOH, a quencher moiety, a reporter moiety, biotin, or a affinity moiety.

### Claim 30 (depends on 1)

The method of claim 1, wherein the biocatalyst is a DNA polymerase.

### Claim 31 (depends on 30)

The method of claim 30, wherein the DNA polymerase has reverse transcriptase activity.

### Claim 32 (depends on 10)

The method of claim 10, wherein the additional nucleotide comprises a terminator nucleotide.