# US 2014/0154683 A1 · Enrichment of Nucleic Acids by Complimentary Capture

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- Number: US 2014/0154683 A1
- Document ID: US20140154683A1
- Status: Published application
- Kind: A1
- Application number: 14/130,007
- Filed: 2012-06-28
- Published: 2014-06-05
- Earliest claimed: 2011-06-29
- Assignees: The Johns Hopkins University
- Inventors: Bert Vogelstein; Kenneth W. Kinzler; Nickolas Papadopoulos; Jian WU
- Web page: https://patentgrep.com/patent/US20140154683A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 2535/122: Massive parallel sequencing
- C12Q 2563/131: the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
- C12Q 2563/149: Particles, e.g. beads
- C12Q 2565/518: characterised by the immobilisation of the nucleic acid sample or target
- 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

Assays can be used to detect mutations found in neoplasms of the pancreas, as well as for other neoplasms and other uses. Nucleic acids can be captured from body fluids such as cyst fluids. Thousands of oligonucleotides can be synthesized in parallel, amplified and ligated together. The ligated products can be further amplified. The amplified, ligated products are used to capture complementary DNA sequences, which can be analyzed, for example by massively parallel sequencing.

## Claims

### Claim 1

A method for enriching a sample comprising nucleic acids for target nucleic acid analytes, comprising the steps of:
synthesizing a set of probes for one or more analytes of interest, wherein the probes are complementary to plus or minus strands of a target nucleic acid analyte, wherein each probe has a common 5′ and 3′ universal priming site;
amplifying the set of probes using primers complementary to the universal priming sites;
ligating the amplified probes to form concatamers;
isothermally amplifying the concatamers;
binding the amplified concatamers to a solid support;
contacting the solid support with the sample comprising nucleic acids under hybridization conditions so that complementary nucleic acids in the sample are captured on the solid support and non-complementary nucleic acids are removed;
eluting captured nucleic acids from the solid support.

### Claim 2

A method for enriching a sample for target nucleic acid analytes, comprising the steps of:
synthesizing a set of probes for one or more analytes of interest, wherein the probes are complementary to plus or minus strands of a target nucleic acid analyte, wherein each probe has a common 5′ and 3′ universal priming site;
amplifying the set of probes using primers complementary to the universal priming sites;
ligating the amplified probes to form concatamers;
isothermally amplifying the concatamers in the presence of biotyinylated nucleotides, such that biotinylated nucleotides are incorporated into the concatamers;
contacting the amplified concatamers with the sample nucleic acids to form a mixture, and subjecting the mixture to hybridization conditions so that complementary nucleic acids in the mixture hybridize to the concatamers;
contacting the mixture with a solid support so that hybridized nucleic acids are captured on the solid support, wherein the solid support comprises avidin or streptavidin;
washing the solid support to remove nucleic acids which do not comprise biotin;
eluting captured nucleic acids from the solid support.

### Claim 3 (depends on 1)

The method of claim 1 further comprising the step of: analyzing nucleotide sequence of the eluted, captured nucleic acids.

### Claim 4 (depends on 3)

The method of claim 3 wherein the analyzing is massively parallel.

### Claim 5 (depends on 2)

The method of claim 2 further comprising: analyzing nucleotide sequence of the eluted, captured nucleic acids.

### Claim 6 (depends on 5)

The method of claim 5 wherein the analyzing is massively parallel.

### Claim 7 (depends on 1)

The method of claim 1 further comprising the step of: amplifying the eluted, captured nucleic acids.

### Claim 8 (depends on 2)

The method of claim 2 further comprising the step of: amplifying the eluted, captured nucleic acids.

### Claim 9 (depends on 1)

The method of claim 1 wherein the set of probes are synthesized on a solid array.

### Claim 10 (depends on 9)

The method of claim 9 wherein the set of probes are cleaved from the solid array to form an aqueous solution comprising the set of probes.

### Claim 11 (depends on 2)

The method of claim 2 wherein the set of probes are synthesized on a solid array.

### Claim 12 (depends on 11)

The method of claim 11 wherein the set of probes are cleaved from the solid array to form an aqueous solution comprising the set of probes.

### Claim 13 (depends on 1)

The method of claim 1 wherein the probes are complementary to both plus and minus strands of a target nucleic acid analyte.

### Claim 14 (depends on 1)

The method of claim 1 wherein probes for one analyte overlap with a register of three displacement.

### Claim 15 (depends on 13)

The method of claim 13 wherein probes for one analyte overlap with a register of three displacement.

### Claim 16 (depends on 1)

The method of claim 1 wherein the solid support is a filter.

### Claim 17 (depends on 1)

The method of claim 1 wherein the solid support is a bead.

### Claim 18 (depends on 2)

The method of claim 2 wherein the probes are complementary to both plus and minus strands of a target nucleic acid analyte.

### Claim 19 (depends on 2)

The method of claim 2 wherein probes for one analyte overlap with a register of three displacement.

### Claim 20 (depends on 18)

The method of claim 18 wherein probes for one analyte overlap with a register of three displacement.

### Claim 21 (depends on 2)

The method of claim 2 wherein the solid support is a filter.

### Claim 22 (depends on 2)

The method of claim 2 wherein the solid support is a bead.