# US 2025/0084455 A1 · Multiplex amplification detection assay

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

- Number: US 2025/0084455 A1
- Document ID: US20250084455A1
- Status: Published application
- Kind: A1
- Application number: 18/732,175
- Filed: 2024-06-03
- Published: 2025-03-13
- Earliest claimed: 2015-10-30
- Assignees: Exact Sciences Corporation
- Applicants: Exact Sciences Corporation
- Inventors: Graham P. Lidgard; Hatim T. Allawi
- Web page: https://patentgrep.com/patent/US20250084455A1

## 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 at position 1

1-22. (canceled)

### Claim 23

A method of analyzing a biological sample from a subject for multiple target nucleic acids in a PCR-flap assay, comprising:
a) providing a DNA sample prepared from a biological sample from a subject, the DNA sample suspected of comprising one or more of at least 3 different target regions, the DNA sample further comprising an endogenous reference DNA and an exogenous process control DNA;
b) treating the DNA sample in an amplification reaction in a PCR-flap assay buffer under conditions wherein one or more of the at least 3 different target regions, if present in the DNA sample, the endogenous reference DNA, and the exogenous process control DNA are amplified to form a pre-amplified mixture, wherein the PCR-flap assay buffer comprises 3-(n-morpholino) propanesulfonic acid (MOPS) buffer and 6 to 10 mM MgCl2,
wherein the amplification reaction comprises PCR amplification reagents comprising:
i) at least 3 different primer pairs for amplifying the at least 3 different target regions, if present in the DNA sample;
ii) a primer pair for amplifying the endogenous reference DNA; and
iii) a primer pair for amplifying the exogenous process control DNA;
c) partitioning the pre-amplified mixture into a plurality of different PCR-flap assay reaction mixtures comprising the PCR-flap assay buffer, each PCR-flap assay reaction mixture comprising an additional amount of one or more primer pairs selected from the at least 3 different primer pairs, an additional amount of the primer pair for amplifying the endogenous reference DNA, and an additional amount of the primer pair for amplifying the exogenous process control DNA; and
d) conducting a plurality of different PCR-flap assays with the PCR-flap assay reaction mixtures, wherein a target region selected from the at least 3 different target regions, if present in the DNA sample at step a), the endogenous reference DNA, and the exogenous process control DNA are amplified and detected in each of the PCR-flap assays.

### Claim 24 (depends on 23)

The method of claim 23, wherein the subject is a human subject.

### Claim 25 (depends on 23)

The method of claim 23, wherein the biological sample is a body fluid.

### Claim 26 (depends on 25)

The method of claim 25, wherein the body fluid comprises plasma.

### Claim 27 (depends on 26)

The method of claim 26, wherein the DNA sample comprises cell-free DNA.

### Claim 28 (depends on 27)

The method of claim 27, wherein the cell-free DNA is less than 200 base pairs in length.

### Claim 29 (depends on 23)

The method of claim 23, wherein the exogenous process control DNA comprises synthetic DNA fragments of fewer than 200 nucleotides in length.

### Claim 30 (depends on 29)

The method of claim 29, wherein the synthetic DNA fragments are double-stranded.

### Claim 31 (depends on 29)

The method of claim 29, wherein the synthetic DNA fragments are methylated.

### Claim 32 (depends on 23)

The method of claim 23, wherein the DNA sample comprises bisulfite-treated DNA.

### Claim 34 (depends on 23)

The method of claim 23, wherein the pre-amplified mixture is diluted with a diluent prior to partitioning.

### Claim 35 (depends on 23)

The method of claim 23, wherein the amplification reaction and/or the plurality of different PCR-flap assay reaction mixtures comprise bulk fish DNA.

### Claim 36 (depends on 23)

The method of claim 23, wherein the PCR-flap assay reaction mixtures comprise a FEN-1 endonuclease.

### Claim 37 (depends on 36)

The method of claim 36, wherein the PCR-flap assay reaction mixtures further comprise:
i) one or more flap oligonucleotide, and
ii) one or more hairpin oligonucleotides, each comprising a region that is complimentary to a portion of one or more flap oligonucleotides.

### Claim 38 (depends on 37)

The method of claim 37, wherein each hairpin oligonucleotide comprises a fluorophore moiety.

### Claim 39 (depends on 23)

The method of claim 23, wherein the amplification reaction of step b) is exposed to thermal cycling conditions limited to fewer than 20 thermal cycles.

### Claim 40 (depends on 23)

The method of claim 23, wherein the amplification reaction of step b) is exposed to thermal cycling conditions limited to 10 or fewer thermal cycles.