# US 10,370,652 B2 · Methods of nucleic acid amplification and sequencing

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- Number: US 10,370,652 B2
- Document ID: US10370652B2
- Status: Granted
- Kind: B2
- Application number: 15/050,345
- Filed: 2016-02-22
- Published: 2016-11-03
- Granted: 2019-08-06
- Earliest claimed: 1998-09-30
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.; Illumina Cambridge Limited
- Inventors: Celine Adessi; Eric Kawashima; Pascal Mayer; Jean-Jacques Mermod; Gerardo Turcatti
- Web page: https://patentgrep.com/patent/US10370652B2

## CPC

- C12N 15/1068: {Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis}
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

Methods for amplification and sequencing of at least one nucleic acid comprising the following steps: (1) forming at least one nucleic acid template comprising the nucleic acid(s) to be amplified or sequenced, wherein said nucleic acid(s) contains at the 5′ end an oligonucleotide sequence Y and at the 3′ end an oligonucleotide sequence Z and, in addition, the nucleic acid(s) carry at the 5′ end a means for attaching the nucleic acid(s) to a solid support; (2) mixing said nucleic acid template(s) with one or more colony primers X, which can hybridize to the oligonucleotide sequence Z and carries at the 5′ end a means for attaching the colony primers to a solid support, in the presence of a solid support so that the 5′ ends of both the nucleic acid template and the colony primers bind to the solid support; (3) performing one or more nucleic acid amplification reactions on the bound template(s), so that nucleic acid colonies are generated and optionally, performing at least one step of sequence determination of one or more of the nucleic acid colonies generated. Solid supports, kits and apparatus for use in these methods.

## Claims

### Claim 1

A method comprising the following steps:
(1) forming at least one first nucleic acid template comprising at least one nucleic acid to be amplified, wherein said at least one nucleic acid contains (a) an oligonucleotide sequence Y and a means for attaching the at least one first nucleic acid template to a solid support at its 5′ end and (b) an oligonucleotide sequence Z at its 3′ end;
(2) mixing said at least one first nucleic acid template with one or more colony primers X, each of which carries a means for attaching one of the one or more colony primers X to the solid support at its 5′ end, in the presence of the solid support such that the 5′ ends of both the at least one first nucleic acid template and the one or more colony primers X bind to the solid support and resulting in at least one bound first template and one or more bound colony primers, wherein at least a subset of the one or more colony primers X can hybridize to the oligonucleotide sequence Z;
(3) performing one or more nucleic acid amplification reactions on the at least one bound first nucleic acid template, so that a first nucleic acid colony is generated, wherein the first nucleic acid colony comprises multiple copies of the at least one bound first nucleic acid template; and
(4) performing at least one step of sequence determination of one or more of the multiple copies of the at least one bound first nucleic acid template while the one or more of the multiple copies of the at least one bound first nucleic acid template is bound to the solid support.

### Claim 2 (depends on 1)

The method of claim 1, wherein the oligonucleotide sequence Z is complementary to the oligonucleotide sequence Y and the oligonucleotide sequence of the one or more colony primers X is the same as the oligonucleotide sequence Y.

### Claim 3 (depends on 1)

The method of claim 1, wherein the one or more colony primers X comprise two different colony primers X, and wherein the two different colony primers X are mixed with said at least one first nucleic acid template in step (2), and wherein the sequence of one of the two different colony primers X is capable of hybridizing to the oligonucleotide sequence Z, and wherein the sequence of the other of the two different colony primers X is the same as the oligonucleotide sequence Y.

### Claim 4 (depends on 1)

The method of claim 1, wherein the step of sequence determination involves incorporation and detection of labelled nucleotides on the one or more of the multiple copies of the at least one bound first nucleic acid template.

### Claim 5 (depends on 4)

The method of claim 4, wherein the method further comprises
(1′) forming additional nucleic acid templates, wherein each of the additional nucleic acid templates contains (a) the oligonucleotide sequence Y and a means for attaching one of the additional nucleic acid templates to the solid support at its 5′ end, and (b) the oligonucleotide sequence Z at its 3′ end;
(2′) mixing each of the additional nucleic acid templates with the one or more colony primers X in the presence of the solid support such that the 5′ ends of both each of the additional nucleic acid templates and the one or more colony primers X bind to the solid support and resulting in bound additional templates and the one or more bound colony primers, wherein at least a subset of the one or more colony primers X can hybridize to the oligonucleotide sequence Z;
(3′) performing one or more nucleic acid amplification reactions on the bound additional nucleic acid templates, so that one or more additional nucleic acid colonies are generated in addition to the first nucleic acid colony, each of the one or more additional nucleic acid colonies comprising multiple copies of one of the bound additional nucleic acid templates, each of the additional nucleic acid templates having a different nucleic acid sequence, and wherein the sequences of the bound additional nucleic acid templates and the sequence of the bound first template are determined simultaneously.

### Claim 6 (depends on 4)

The method of claim 4, further comprising an additional step of visualising the first colony.

### Claim 7 (depends on 6)

The method of claim 6, wherein the step of visualising the first colony involves the use of a labelled or unlabelled nucleic acid probe.

### Claim 8 (depends on 1)

The method of claim 1, wherein said 5′ ends of both the at least one first nucleic acid template and the one or more colony primers X are covalently bound to the solid support.

### Claim 9 (depends on 8)

The method of claim 8, wherein the at least one first nucleic acid template and the one or more colony primers X comprise a chemically modifiable functional group that covalently binds the at least one first nucleic acid template and the one or more colony primers X to the solid support.

### Claim 10 (depends on 9)

The method of claim 9, wherein said chemically modifiable functional group is a phosphate group, a carboxylic or aldehyde moiety, a thiol, a hydroxyl, a dimethoxytrityl (DMT), or an amino group.

### Claim 11 (depends on 10)

The method of claim 10, wherein said chemically modifiable functional group is an amino group.

### Claim 12 (depends on 1)

The method of claim 1, wherein said solid support is selected from the group consisting of latex beads, dextran beads, polystyrene and polypropylene surfaces, polyacrylamide gel, gold surfaces, and silicon wafers.

### Claim 13 (depends on 1)

The method of claim 1, wherein the solid support is glass.

### Claim 14 (depends on 5)

The method of claim 5, wherein the densities of the first nucleic acid colony and the additional nucleic acid colonies on the solid support are 10,000/mm2 to 100,000/mm2.

### Claim 15 (depends on 1)

The method of claim 1, wherein the density of the one or more bound colony primers X on the solid support is at least 1 fmol/mm2.

### Claim 16 (depends on 1)

The method of claim 1, wherein the density of the at least one bound first nucleic acid template in the first nucleic acid colony is 10,000/mm2 to 100,000/mm2.