# US 2019/0376136 A1 · Conformal probes and methods for sequencing nucleic acids

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- Number: US 2019/0376136 A1
- Document ID: US20190376136A1
- Status: Published application
- Kind: A1
- Application number: 16/352,731
- Filed: 2019-03-13
- Published: 2019-12-12
- Earliest claimed: 2010-06-18
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Molly He; Cheng-Yao Chen; Eric Kool; Mostafa Ronaghi; Michael Previte; Rigo Pantoja
- Web page: https://patentgrep.com/patent/US20190376136A1

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- 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

This disclosure provides a method of determining a sequence of nucleotides for a nucleic acid template. The method can include the steps of contacting the nucleic acid template with a conformationally labeled polymerase and at least four different nucleotide species under conditions wherein the conformationally labeled polymerase catalyzes sequential addition of the nucleotide species to form a nucleic acid complement of the nucleic acid template, wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase and wherein the rate or time duration for the conformational signal change is distinguishable for each different nucleotide species; detecting a series of changes in the signal from the conformationally labeled polymerase under the conditions; and determining the rates or time durations for the changes in the signal, thereby determining the sequence of nucleotides for the nucleic acid template.

## Claims

### Claim 1

A method of determining a sequence of nucleotides for a nucleic acid sample, comprising
providing an array of nucleic acid templates, wherein the nucleic acid templates comprise nucleotide sequence fragments of the nucleic acid sample;
contacting the array of nucleic acid templates with;
(i) a conformationally labeled polymerase, wherein the conformationally labeled polymerase comprises a finger domain labeled with a first fluorescent label and a thumb domain labeled with a second label, wherein the first fluorescent label and second label are FRET pairs or wherein the second label is a quencher; and
(ii) at least four different nucleotide species under conditions wherein the conformationally labeled polymerases catalyze sequential addition of the nucleotide species to form nucleic acid complements of the nucleic acid templates, wherein the different nucleotide species comprise at least one different non-natural nucleotide analog, wherein the sequential addition of each different nucleotide species produces a conformational fluorescent signal change from the conformationally labeled polymerase and wherein the rate or time duration for the conformational fluorescent signal change is distinguishable for each different nucleotide species;
detecting a series of changes in the signal from the conformationally labeled polymerase under the conditions and at individual locations of the array; and
determining the rates or time durations for the changes in the signal at the individual locations of the array, thereby determining the sequence of nucleotides for the nucleic acid sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the providing the array comprises amplifying portions of the nucleic acid sample to produce the nucleotide sequence fragments.

### Claim 3 (depends on 1)

The method of claim 1, wherein the providing the array comprises adding a universal sequence to the nucleotide sequence fragments to form tailed fragments and hybridizing the tailed fragments to oligonucleotides, wherein the oligonucleotides are attached to the array and wherein the oligonucleotides are complementary to the universal sequence.

### Claim 4 (depends on 1)

The method of claim 1, further comprising contacting the array of nucleic acid templates with conformationally labeled exonucleases, wherein the conformationally labeled exonuclease comprises a finger domain labeled with a second fluorescent label and a thumb domain labeled with a third label, wherein the second fluorescent label and third label are FRET pairs and wherein the FRET pairs are different from the FRET pairs of the conformationally labeled polymerase or wherein the third label is a quencher; under conditions wherein the conformationally labeled exonucleases catalyze sequential removal of nucleotide species from the nucleic acid templates, wherein the sequential removal of each different nucleotide species produces a conformational signal change from each of the conformationally labeled exonucleases and wherein the rate or time duration for the conformational signal change is distinguishable for at least one different nucleotide species that is removed;
detecting a series of changes in the signal from the conformationally labeled exonuclease under the conditions and at the individual locations of the array; and determining the rates or time durations for the changes in the signal for the series of changes in the signal from the conformationally labeled exonuclease.

### Claim 5 (depends on 4)

The method of claim 4, wherein the rate or time duration for the conformational signal change is distinguishable for only one of four different nucleotide species that are removed.

### Claim 6 (depends on 4)

The method of claim 4, wherein the rates or time durations for the conformational signal change are distinguishable for four of four different nucleotide species that are removed.

### Claim 7 (depends on 4)

The method of claim 4, wherein the at least one different nucleotide species that is removed comprises a modified alpha-phosphate moiety.

### Claim 8 (depends on 7)

The method of claim 7, wherein the modified alpha-phosphate moiety comprises a moiety other than oxygen covalently attached to the alpha phosphate.

### Claim 9 (depends on 1)

The method of claim 1, wherein the at least one nucleotide species comprise at least four different non-natural nucleotide analogs.

### Claim at position 10

10-13. (canceled)

### Claim 14 (depends on 1)

The method of claim 1, wherein the conformational signal change comprises increased FRET, decreased FRET, increased fluorescence quenching or decreased fluorescence quenching.

### Claim 15 (depends on 1)

The method of claim 1, wherein the nucleic acid templates are individually resolved from each other.

### Claim 16 (depends on 1)

The method of claim 1, wherein the nucleic acid templates are present in colonies and individual colonies comprise a plurality of templates that are the same species.

### Claim 17 (depends on 16)

The method of claim 16, wherein the method comprises determining a sequence of nucleotides for a plurality of templates in the colony.

### Claim 18 (depends on 16)

The method of claim 16, wherein the at least one non-natural nucleotide analog comprises a reversible terminator moiety.

### Claim 19 (depends on 1)

The method of claim 1, wherein the at least one nucleotide species comprise at least one caged nucleotide.

### Claim 20

A method of determining nucleotide sequences, comprising
(a) providing an array of different nucleic acid templates;
(b) providing a mixture of nucleotide species, the mixture comprising
(i) at least four different nucleotide species,
(ii) at least one of the four different nucleotide species comprising a reversible terminator moiety, and
(iii) at least two of the four different nucleotide species comprising an extendible 3′ hydroxyl moiety;
(c) contacting the array of nucleic acid templates with conformationally labeled polymerases, wherein the conformationally labeled polymerase comprises a finger domain labeled with a first fluorescent label and a thumb domain labeled with a second label, wherein the first fluorescent label and second label are FRET pairs or wherein the second label is a quencher; and the mixture of nucleotide species under conditions wherein the conformationally labeled polymerases catalyze sequential addition of the nucleotide species to form nucleic acid complements of the nucleic acid templates,
wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase,
wherein the rate or time duration for the conformational signal change is distinguishable for the at least two nucleotide species comprising the extendible 3′ hydroxyl moiety, and
wherein a plurality of the nucleic acid complements incorporate the at least one nucleotide species that comprises the reversible terminator moiety;
(d) removing the reversible terminator moiety;
(e) detecting a series of changes in the signal from the conformationally labeled polymerase at individual locations of the array; and
(f) determining the sequence of nucleotides for the nucleic acid sample from the series of changes in the signal from the conformationally labeled polymerase.

### Claim 21 (depends on 20)

The method of claim 20, wherein at least one of the four different nucleotide species is present at a substantially lower concentration than the concentration of any other nucleotide species in the mixture, whereby the nucleotide that is present at the substantially lower concentration produces a distinguishable rate or time duration for the conformational signal change.

### Claim 22 (depends on 20)

The method of claim 20, further comprising repeating steps (c), (d) and (e) several times.