# US 2019/0376147 A1 · Compositions and methods for detecting a mutant variant of a polynucleotide

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

- Number: US 2019/0376147 A1
- Document ID: US20190376147A1
- Status: Published application
- Kind: A1
- Application number: 16/480,535
- Filed: 2018-01-23
- Published: 2019-12-12
- Earliest claimed: 2017-01-24
- Assignees: The Broad Institute, Inc.
- Applicants: The Broad Institute, Inc.
- Inventors: Edward F. Fritsch
- Web page: https://patentgrep.com/patent/US20190376147A1

## CPC

- C12Q 1/6858: Allele-specific amplification
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/156: Polymorphic or mutational markers
- 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

In one aspect, the invention features a combination of oligonucleotides comprising a forward primer oligonucleotide and a blocking oligonucleotide. The forward primer oligonucleotide has a 3′ end region, where the 3′ end region includes a portion complementary to a mutation positioned in a region within a polynucleotide. The blocking oligonucleotide contains a blocking moiety and has a 5′ end region, where the 5′ end region includes a portion complementary to a wild-type sequence of the region corresponding to the position of the mutation. In other aspects, the invention provides kits including the combination of primer oligonucleotides and methods of using the oligonucleotides to detect a mutation in a polynucleotide.

## Claims

### Claim at position 1

1-74. (canceled)

### Claim 75

A method of detecting a cancer cell and/or polynucleotide associated with cancer in a sample obtained from a subject, the method comprising
(a) contacting a first polynucleotide strand of a double stranded polynucleotide with a first primer having a 3′ end region, wherein the 3′ end region comprises a portion complementary to a mutation positioned within a region of a polynucleotide, and a blocking oligonucleotide comprising a blocking moiety and having a 5′ end region, wherein the 5′ end region comprises a portion complementary to a wild-type sequence corresponding to the region comprising the mutation within the first polynucleotide strand, wherein the first primer and the blocking oligonucleotide compete for binding to the region of the first polynucleotide strand encompassing the mutation, and
(b) contacting the second polynucleotide strand or the first polynucleotide strand of the double stranded polynucleotide with a second primer comprising a sequence substantially complementary to a sequence on the second strand of the polynucleotide or the first polynucleotide strand that is 5′ of the region corresponding to where the first primer binds the first polynucleotide strand; and
(c) detecting an amplicon in the sample, wherein detection of an amplicon indicates the presence of a cancer cell and/or polynucleotide associated with cancer in the sample.

### Claim 76

(canceled)

### Claim 77 (depends on 75)

The method of claim 75, wherein the mutation is at least one insertion or deletion (indels).

### Claim 78 (depends on 75)

The method of claim 75, wherein the blocking moiety is positioned at the 3′end of the blocking oligonucleotide and prevents polymerase extension.

### Claim 79 (depends on 78)

The method of claim 78, wherein the blocking moiety is selected from the group consisting of 3′-phosphate; 2′, 3′ dideoxyadenosine (2,3ddA); 2′,3′ dideoxycytosine (2,3ddC), 2′,3′ dideoxythymidine (2,3ddT), 2′,3′ dideoxyguanosine (2,3ddG), 3′-deoxycytidine, and 3′-spacer C3.

### Claim 80 (depends on 75)

The method of claim 75, wherein the region of the polynucleotide comprising the mutation comprises up to about 30 nucleotides immediately upstream and/or downstream of the position of the mutation.

### Claim 81 (depends on 75)

The method of claim 75, wherein the portion of the first primer complementary to the mutation comprises the last 1, last 2, last 3, last 4, last 5, last 6, last 7, last 8, last 9, or last 10 nucleotides positioned at the 3′ end of the first primer.

### Claim 82 (depends on 75)

The method of claim 75, wherein the portion of the blocking oligonucleotide complementary to the wild-type sequence of the region of the polynucleotide comprising the mutation comprises the first 1, first 2, first 3, first 4, first 5, first 6, first 7, first 8, first 9, or first 10 nucleotides positioned at the 5′ end of the blocking oligonucleotide.

### Claim 83 (depends on 75)

The method of claim 75, wherein each of the oligonucleotides is at least about 15 to at least about 30 nucleotides in length.

### Claim 84 (depends on 75)

The method of claim 75, wherein the mutation is associated with cancer, wherein the cancer is selected from the group consisting of biliary tract cancer, bladder cancer, glioblastoma, medulloblastoma, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasm, acute lymphocytic, myelogenous leukemia, multiple myeloma, AIDS-associated leukemia, adult T-cell leukemia, lymphoma, intraepithelial neoplasm, Bowen's disease, Paget's disease, liver cancer, lung cancer, Hodgkin's disease, lymphocytic lymphoma, neuroblastoma, oral cancer, squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, osteosarcoma, skin cancer, melanoma, Kaposi's sarcoma, basocellular cancer, testicular cancer, thyroid cancer, and renal cancer.

### Claim 85

(canceled)

### Claim 86 (depends on 75)

The method of claim 75, wherein if the region corresponding to the position of the mutation has the wild-type sequence, the blocking oligonucleotide substantially inhibits amplification of at least a portion of the polynucleotide.

### Claim 87 (depends on 75)

The method of claim 75, wherein if the region corresponding to the position of the mutation has a mutation, the blocking oligonucleotide does not substantially inhibit amplification of at least a portion of the polynucleotide.

### Claim 88 (depends on 75)

The method of claim 75, wherein the sample is a blood sample.

### Claim 89 (depends on 75)

The method of claim 75, wherein the subject is human.

### Claim 90 (depends on 75)

The method of claim 75, wherein the cancer cell is a metastatic cancer cell or a leukemic cell.

### Claim at position 18

91-114. (canceled)

### Claim 115 (depends on 75)

The method of claim 75, wherein the first primer has a sequence identical to a 22-nucleotide region about 1 kilobase (kb) upstream of the region on the polynucleotide strand that is complementary to the second primer.

### Claim 116 (depends on 78)

The method of claim 78, wherein the 3′end of the blocking oligonucleotide cannot serve as a substrate for DNA polymerase.

### Claim 117 (depends on 88)

The method of claim 88, wherein the sample comprises mutant circulating tumor DNAs (ctDNA) from dead tumor circulating tumor cells (CTCs) present among a far excess of wild-type DNA from normal cells.

### Claim 118 (depends on 75)

The method of claim 75, wherein the amplicon is 13 nucleotides shorter when unblocked blocking primer is bound.

### Claim 119 (depends on 75)

The method of claim 75, wherein the first primer is completely bound to the template, and the blocking oligonucleotide is unbound or displaced by the first primer during branch migration and/or strand displacement.

### Claim 120 (depends on 75)

The method of claim 75, wherein the first primer, second primer, or blocking oligonucleotide are synthetically modified.