# US 11,299,766 B2 · Multiplex amplification detection assay

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- Number: US 11,299,766 B2
- Document ID: US11299766B2
- Status: Granted
- Kind: B2
- Application number: 16/888,375
- Filed: 2020-05-29
- Published: 2020-09-17
- Granted: 2022-04-12
- Earliest claimed: 2015-10-30
- Assignees: Exact Sciences Corporation
- Applicants: Exact Sciences Corporation
- Inventors: Graham P. Lidgard; Hatim Allawi
- Web page: https://patentgrep.com/patent/US11299766B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2600/154: Methylation markers
- C12Q 2600/166: Oligonucleotides used as internal standards, controls or normalisation probes
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein is technology relating to the amplification-based detection of bisulfite-treated DNAs and particularly, but not exclusively, to methods and compositions for multiplex amplification of low-level sample DNA prior to further characterization of the sample DNA. The technology further provides methods for isolating DNA from blood or blood product samples, e.g., plasma samples.

## Claims

### Claim 1

A method of analyzing a sample for multiple target nucleic acids in a PCR-flap assay, comprising:
a) providing a sample comprising DNA suspected of containing one or more of a plurality of different target regions;
b) treating the sample in an amplification reaction mixture comprising a PCR-flap assay buffer comprising 6 to 10 mM Mg′ under conditions wherein the different target regions, if present in the sample, are amplified to form a pre-amplified mixture;
c) partitioning the pre-amplified mixture into a plurality of different PCR-flap assay reaction mixtures comprising the PCR-flap assay buffer; and
d) conducting a plurality of PCR-flap assays with the PCR-flap assay reaction mixtures, wherein the different target regions, if present in the sample at step a), are amplified and detected in the PCR-flap assay reaction mixtures.

### Claim 2 (depends on 1)

The method of claim 1, wherein the DNA is from a human subject.

### Claim 3 (depends on 1)

The method of claim 1, wherein the sample is prepared from a body fluid.

### Claim 4 (depends on 1)

The method of claim 1, wherein the body fluid comprises plasma.

### Claim 5 (depends on 1)

The method of claim 1, wherein the DNA comprises bisulfite-treated DNA.

### Claim 6 (depends on 1)

The method of claim 1, wherein the sample is prepared from cell-free DNA isolated from plasma.

### Claim 7 (depends on 1)

The method of claim 1, wherein the amplification reaction comprises at least 3 different primer pairs for amplifying at least 3 different target regions, if present in the sample.

### Claim 8 (depends on 1)

The method of claim 1, wherein the amplification reaction comprises at least 4 different primer pairs for amplifying at least 4 different target regions, if present in the sample.

### Claim 9 (depends on 1)

The method of claim 1, wherein the pre-amplified mixture is diluted with a diluent prior to partitioning.

### Claim 10 (depends on 1)

The method of claim 1, wherein the amplification reaction mixture and/or the plurality of different PCR-flap assay reaction mixtures comprise bulk fish DNA.

### Claim 11 (depends on 1)

The method of claim 1, wherein the PCR-flap assay reaction mixtures comprise a FEN-1 endonuclease.

### Claim 12 (depends on 1)

The method of claim 1, wherein the PCR-flap assay buffer comprises MgCl2.

### Claim 13 (depends on 1)

The method of claim 1, wherein the amplification reaction mixture comprises at least 2 different primer pairs for amplifying the at least 2 different target regions, and wherein each PCR-flap assay reaction mixture comprises an additional amount of a primer pair selected from the at least 2 different primer pairs.

### Claim 14 (depends on 13)

The method of claim 13, wherein each PCR-flap assay reaction mixture further comprises:
i) a flap oligonucleotide, and
ii) a hairpin oligonucleotide comprising a region that is complimentary to a portion of the flap oligonucleotide.

### Claim 15 (depends on 1)

The method of claim 1, wherein the amplification reaction mixture of step b) is exposed to thermal cycling conditions limited to fewer than 20 thermal cycles.

### Claim 16 (depends on 15)

The method of claim 15, wherein the hairpin oligonucleotide comprises a fluorophore moiety.

### Claim 17 (depends on 1)

The method of claim 1, wherein the plurality of different target regions comprises a reference target region.

### Claim 18 (depends on 17)

The method of claim 17, wherein said reference target region comprises β-actin or ZDHHC1.

### Claim 19 (depends on 1)

The method of claim 1, wherein the sample comprises a DNA process control.

### Claim 20 (depends on 19)

The method of claim 19, wherein the DNA process control comprises a zebrafish RASSF1 sequence.