# US 11,193,168 B2 · Multiplex amplification detection assay II

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- Number: US 11,193,168 B2
- Document ID: US11193168B2
- Status: Granted
- Kind: B2
- Application number: 16/789,279
- Filed: 2020-02-12
- Published: 2020-08-06
- Granted: 2021-12-07
- Earliest claimed: 2017-12-13
- Assignees: Exact Sciences Development Company, LLC
- Applicants: Exact Sciences Development Company, LLC
- Inventors: Hatim T. Allawi; William G. Weisburg; Graham P. Lidgard; Michael W. Kaiser; Abram M. Vaccaro; Gracie Shea
- Web page: https://patentgrep.com/patent/US11193168B2

## CPC

- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6827: for detection of mutation or polymorphism
- C01B 17/48: Sulfur dioxide; Sulfurous acid
- C12N 15/65: using markers (enzymes used as markers C12N15/52)
- C12N 2310/122: Hairpin
- C12Q 2521/301: Endonuclease
- C12Q 2531/113: PCR
- C12Q 2600/16: Primer sets for multiplex assays
- 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
- C01B: NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; {METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C}
- 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, comprising:
(a) amplifying a sample comprising bisulfite-treated DNA by PCR in a single reaction using a plurality of different primer pairs to produce a pre-amplified mixture;
(b) partitioning the pre-amplified mixture into a plurality of different detection assay reaction mixtures, wherein each detection assay reaction mixture comprises a portion of said pre-amplified mixture; and
(c) conducting a plurality of detection assays on the detection assay reaction mixtures, wherein the detection assays are PCR-flap assays that employ flap oligonucleotides that have a target-specific region of at least 13 bases in length and an additional amount of a primer pair selected from said plurality of different primer pairs of step (a), wherein the primer pair selected is not a nested primer pair or a semi-nested primer pair.

### Claim 2 (depends on 1)

The method of claim 1, wherein one or more of the flap oligonucleotides used in (c) has a target-specific region comprising one or more nucleotides that are capable of making non-Watson-Crick base pairs.

### Claim 3 (depends on 1)

The method of claim 1, wherein one or more of the flap oligonucleotides used in (c) has a target-specific region having a length in the range of 13 to 30 bases.

### Claim 4 (depends on 1)

The method of claim 1, wherein the one or more of the flap oligonucleotides used in (c) has a target-specific region that has a Tm in the range of 60° C. to 70° C. and the detection assays comprise a denaturation step at least 90° C., an annealing step at a temperature that is in the range of 60° C. to 70° C., and an extension step at a temperature in the range of 65° C. to 75° C.

### Claim 5 (depends on 1)

The method of claim 1, wherein said bisulfite treated DNA is from a human subject.

### Claim 6 (depends on 5)

The method of claim 5, wherein said sample is prepared from a body fluid.

### Claim 7 (depends on 6)

The method of claim 6, wherein said body fluid comprises plasma.

### Claim 8 (depends on 7)

The method of claim 7, wherein the sample is prepared from cell-free DNA isolated from plasma.

### Claim 9 (depends on 8)

The method of claim 8, wherein said cell-free DNA is less than 200 base pairs in length.

### Claim 10 (depends on 8)

The method of claim 8, wherein said cell-free DNA is isolated from said plasma by a method comprising:
a) combining the plasma sample with:
i) protease; and
ii) a first lysis reagent, said first lysis reagent comprising
guanidine thiocyanate; and
non-ionic detergent;
to form a mixture wherein proteins are digested by said protease;
b) to the mixture of step a) adding
iii) silica particles, and
iv) a second lysis reagent, said second lysis reagent comprising:
guanidine thiocyanate;
non-ionic detergent; and
isopropyl alcohol;
under conditions wherein DNA is bound to said silica particles;
c) separating silica particles with bound DNA from the mixture of b);
d) to the separated silica particles with bound DNA adding a first wash solution, said first wash solution comprising guanidine hydrochloride or guanidine thiocyanate and ethyl alcohol;
e) separating the silica particles with bound DNA from said first wash solution;
f) to the separated silica particles with bound DNA adding a second wash solution, said second wash solution comprising a buffer and ethyl alcohol;
g) separating washed silica particles with bound DNA from said second wash solution; and
h) eluting DNA from the washed silica particles with bound DNA.

### Claim 11 (depends on 10)

The method of claim 10, wherein said protease is Proteinase K.

### Claim 12 (depends on 6)

The method of claim 6, wherein the sample is prepared from at least one mL of bodily fluid.

### Claim 13 (depends on 1)

The method of claim 1, wherein the pre-amplified mixture is partitioned into at least 4 detection assay reaction mixtures.