# US 2017/0283794 A1 · Methods for selecting and amplifying polynucleotides

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2017/0283794 A1
- Document ID: US20170283794A1
- Status: Published application
- Kind: A1
- Application number: 15/489,549
- Filed: 2017-04-17
- Published: 2017-10-05
- Earliest claimed: 2008-03-10
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Andrea Sabot; Roberto Rigatti; Min-Jui Richard Shen
- Web page: https://patentgrep.com/patent/US20170283794A1

## 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}
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6844: Nucleic acid amplification reactions
- 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)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- 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 invention provides methods for generating a nucleic acid library. Target-specific primer-probes are hybridized to a target nucleic acid fragment to create a hybridization product. Each of the target-specific primer-probes comprises a target-specific sequence and a first adaptor sequence. The target nucleic acid fragment comprises a target genomic region of interest, wherein said target genomic region of interest comprises an exon of a gene and the target-specific primer-probes are tiled across the exon of the gene. The target nucleic acid fragment further comprises a second adaptor sequence different from said first adaptor sequence. Following hybridization, the target-specific primer-probes are extended to create double-stranded extension products and further amplified, thereby generating the nucleic acid library.

## Claims

### Claim 1

(canceled)

### Claim 2

A method for generating a nucleic acid library, the method comprising:
a) hybridizing target-specific primer-probes to a target nucleic acid fragment to create a hybridization product;
b) extending said target-specific primer-probes to create double-stranded extension products; and
c) amplifying said extension products, thereby generating the nucleic acid library;
wherein each of said target-specific primer-probes comprises a target-specific sequence and a first adaptor sequence; said target nucleic acid fragment comprises a target genomic region of interest comprising an exon of a gene; said target-specific primer-probes are tiled across said exon of said gene; and said target nucleic acid fragment comprises a second adaptor sequence different from said first adaptor sequence.

### Claim 3 (depends on 2)

The method of claim 2, wherein said target nucleic acid fragment comprises DNA.

### Claim 4 (depends on 2)

The method of claim 2, wherein said target nucleic acid fragment comprises RNA.

### Claim 5 (depends on 2)

The method of claim 2, wherein said hybridization, extension and said amplification steps are performed directly inside a next-generation DNA sequencer.

### Claim 6 (depends on 2)

The method of claim 2, wherein said target-specific primer-probes are selectively hybridizable to said target genomic region of interest.

### Claim 7 (depends on 2)

The method of claim 2, wherein said amplification comprises bridge amplification.

### Claim 8 (depends on 2)

The method of claim 2, further comprising sequencing said amplified extension products.

### Claim 9 (depends on 8)

The method of claim 8, wherein said sequencing comprises use of a parallel sequencing platform.

### Claim 10 (depends on 9)

The method of claim 9, wherein said first adaptor sequence comprises a sequencing platform-specific sequence for binding to a solid support of said sequencing platform.

### Claim 11 (depends on 2)

The method of claim 2, wherein said first adaptor sequence comprises a binding site for a sequencing primer.

### Claim 12 (depends on 2)

The method of claim 2, further comprising ligating said second adaptor sequence to said target nucleic acid fragment.

### Claim 13 (depends on 12)

The method of claim 12, wherein said second adaptor sequence is ligated to one end but not both ends of said target nucleic acid fragment.

### Claim 14 (depends on 2)

The method of claim 2, wherein said second adaptor sequence comprises an index sequence.

### Claim 15 (depends on 14)

The method of claim 14, wherein said index sequence allows a source of said target nucleic acid fragment to be identified.

### Claim 16 (depends on 2)

The method of claim 2, further comprising hybridizing a target-specific primer-probe to a sequence that flanks said exon.

### Claim 17 (depends on 2)

The method of claim 2, further comprising hybridizing said target-specific primer-probes to each strand of said exon.

### Claim 18 (depends on 2)

The method of claim 2, further comprising performing targeted resequencing of said gene in a plurality of samples.

### Claim 19 (depends on 2)

The method of claim 2, further comprising hybridizing a plurality of target-specific primer-probes to a plurality of target nucleic acid fragments.

### Claim 20 (depends on 2)

The method of claim 2, wherein said gene is a ribosomal gene.

### Claim 21 (depends on 2)

The method of claim 2, wherein said ribosomal gene is 16S or 18S rRNA gene.