# US 11,999,996 B2 · Polynucleotide sequence detection method

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- Number: US 11,999,996 B2
- Document ID: US11999996B2
- Status: Granted
- Kind: B2
- Application number: 17/181,445
- Filed: 2021-02-22
- Published: 2021-06-17
- Granted: 2024-06-04
- Earliest claimed: 2018-07-19
- Assignees: Biofidelity Ltd
- Applicants: Biofidelity Ltd
- Inventors: Barnaby Balmforth; Cameron Frayling; Ana Silva-Weatherley; Magdalena Stolarek-Januszkiewicz
- Web page: https://patentgrep.com/patent/US11999996B2

## 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

A method of detecting a target polynucleotide sequence in a given nucleic acid analyte characterised by the steps of: a. annealing the analyte to a single-stranded probe oligonucleotide A0 to create a first intermediate product which is at least partially double-stranded and in which the 3′ end of A0 forms a double-stranded complex with the analyte target sequence; b. pyrophosphorolysing the first intermediate product with a pyrophosphorolysing enzyme in the 3′-5′ direction from the 3′ end of A0 to create partially digested strand A1 and the analyte; c. (i) annealing A1 to a single-stranded trigger oligonucleotide B and extending the A1 strand in the 5′-3′ direction against B; or (ii) circularising A1 through ligation of its 3′ and 5′ ends; or (iii) ligating the 3′ end of A1 to the 5′ end of a ligation probe oligonucleotide C; in each case to create an oligonucleotide A2; d. priming A2 with at least one single-stranded primer oligonucleotide and creating multiple copies of A2, or a region of A2; and e. detecting a signal derived from the multiple copies and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte.

## Claims

### Claim 1

A kit comprising:
(a) a single-stranded probe oligonucleotide A0 comprising a 3′ end that is perfectly complementary to a target polynucleotide sequence, a priming sequence, an identification region, and a 5′ end that is resistant to 5′-3′ exonuclease digestion;
(b) a ligase;
(c) a pyrophosphorolysing enzyme; and
(d) an inorganic source of pyrophosphate ions.

### Claim 2

A reaction mixture comprising:
(a) a single-stranded probe oligonucleotide A0 comprising a 3′ end that is perfectly complementary to a target polynucleotide sequence, a priming sequence, an identification region, a 5′ phosphate, and a 5′ end that is resistant to 5′-3′ exonuclease digestion;
(b) a ligase;
(c) a pyrophosphorolysing enzyme; and
(d) an inorganic source of pyrophosphate ions.

### Claim 3 (depends on 2)

The reaction mixture of claim 2, wherein the source of pyrophosphate ions includes an excess amount.

### Claim 4 (depends on 2)

The reaction mixture of claim 2, further comprising a splint oligonucleotide.

### Claim 5 (depends on 4)

The reaction mixture of claim 4, wherein the splint oligonucleotide comprises a first region complementary to the 3′ end of an oligonucleotide strand A1 and a second region complementary to the 5′ end of A1 and the splint is unable to undergo extension against A1 by virtue of a mismatch between the 3′ end of the splint and the corresponding region of A1.

### Claim 6 (depends on 2)

The reaction mixture of claim 2, further comprising the target polynucleotide sequence.

### Claim 7 (depends on 2)

The reaction mixture of claim 2, further comprising a 5′-3′ exonuclease.

### Claim 8 (depends on 2)

The reaction mixture of claim 2, further comprising a phosphatase.

### Claim 9 (depends on 8)

The reaction mixture of claim 8, wherein the phosphatase is apyrase.

### Claim 10 (depends on 2)

The reaction mixture of claim 2, wherein the reaction mixture comprises a plurality of single-stranded probe oligonucleotides A0, wherein each oligonucleotide A0 has the same priming sequence.

### Claim 11 (depends on 10)

The reaction mixture of claim 10, wherein the plurality of single-stranded probe oligonucleotides A0 comprises a first oligonucleotide A0 and a second oligonucleotide A0, wherein the first and second oligonucleotides A0 comprise the same priming sequence, the first A0 oligonucleotide comprises a 3′ end that is perfectly complementary to a first target polynucleotide sequence, and the second A0 nucleotide comprises a 3′ end that is perfectly complementary to a second target polynucleotide sequence.

### Claim 12 (depends on 2)

The reaction mixture of claim 2, wherein the reaction mixture comprises a plurality of single-stranded oligonucleotides A0, wherein each oligonucleotide A0 includes two or more priming sequences.

### Claim 13 (depends on 2)

The reaction mixture of claim 2, wherein the single-stranded probe oligonucleotide A0 is complementary to a mutation in the EGFR gene.

### Claim 14 (depends on 13)

The reaction mixture of claim 13, wherein the reaction mixture comprises one or more of:
an oligonucleotide A0 complementary to the T790M mutation of the EGFR gene;
an oligonucleotide A0 complementary to the C797S mutation of the EGFR gene;
an oligonucleotide A0 complementary to the L861Q mutation of the EGFR gene;
an oligonucleotide A0 complementary to the L858R mutation of the EGFR gene;
an oligonucleotide A0 complementary to a deletion in exon 19 of the EGFR gene;
an oligonucleotide A0 complementary to an insertion in exon 19 of the EGFR gene; and/or
an oligonucleotide A0 complementary to an insertion in exon 20 of the EGFR gene.

### Claim 15 (depends on 2)

The reaction mixture of claim 2, further comprising a blocking oligonucleotide including a 3′ end region at least partially mismatched to the target polynucleotide sequence, wherein the presence of the blocking oligonucleotide promotes annealing of oligonucleotide A0 only to the target polynucleotide sequence.

### Claim 16 (depends on 2)

The reaction mixture of claim 2, further comprising a blocking oligonucleotide that has a 3′ end region at least partially mismatched to the target polynucleotide sequence, wherein the presence of the blocking oligonucleotide promotes annealing of oligonucleotide A0 only to the target polynucleotide sequence.

### Claim 17 (depends on 2)

The reaction mixture of claim 2, further comprising:
a splint oligonucleotide comprising a first region complementary to the 3′ end of an oligonucleotide strand A1 and a second region complementary to the 5′ end of A1, wherein the splint oligonucleotide is unable to undergo extension against A1 by virtue of a mismatch between the 3′ end of the splint and the corresponding region of A1;
the target polynucleotide sequence, wherein the 3′ end of oligonucleotide A0 is perfectly complementary to the target polynucleotide sequence;
a 5′-3′ exonuclease; and
a phosphatase.

### Claim 18 (depends on 2)

The reaction mixture of claim 2, further comprising:
the target polynucleotide sequence, wherein the 3′ end of oligonucleotide A0 is perfectly complementary to the target polynucleotide sequence;
a 5′-3′ exonuclease; and
a phosphatase.