# US 11,306,350 B2 · Primers, compositions, and methods for nucleic acid sequence variation detection > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,306,350 B2 - Document ID: US11306350B2 - Status: Granted - Kind: B2 - Application number: 16/384,262 - Filed: 2019-04-15 - Published: 2020-04-02 - Granted: 2022-04-19 - Earliest claimed: 2018-09-30 - Assignees: Maccura Biotechnology Co., Ltd. - Applicants: Maccura Biotechnology Co., Ltd. - Inventors: Yuhang Zhao; Shufang Wang; Zhiqi Ge - Web page: https://patentgrep.com/patent/US11306350B2 ## CPC - C12Q 1/6851: Quantitative amplification - C12Q 1/6858: Allele-specific amplification - C12Q 1/6827: for detection of mutation or polymorphism - 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 Specific, accurate, and cost effective primers for performing digital PCR, compositions and kits containing the primer and methods for using and making the same are useful for detecting nucleic acid mutations. A primer useful as a first forward primer in performing digital PCR to detect a target nucleic acid in a sample, includes: a detection portion located upstream to a target sequence binding portion, and including a second forward primer binding portion having a sequence substantially complementary to a second forward primer, and a probe binding portion downstream to the second forward primer binding portion having a sequence substantially complementary to a probe; the target sequence binding portion includes a mismatch portion having a sequence not complementary to the target nucleic acid, and an amplification determinant portion downstream to the mismatch portion having a sequence complementary to a gene allele or a variant thereof encoded by the target nucleic acid. ## Claims ### Claim 1 A component configured to perform digital PCR, comprising a primer as a first forward primer to detect a target nucleic acid in a sample, the primer comprising: a target sequence binding portion; and a detection portion located upstream to the target sequence binding portion, wherein said detection portion comprises: a second forward primer binding portion having a sequence substantially complementary to a second forward primer, and a probe binding portion downstream to the second forward primer binding portion having a sequence substantially complementary to a probe; and said target sequence binding portion comprises: a mismatch portion having a sequence not complementary to the target nucleic acid, and an amplification determinant portion different from, and downstream, to the mismatch portion, the amplification determinant portion having a sequence complementary to a gene allele or a variant thereof encoded by the target nucleic acid; wherein there are no other bases downstream of the amplification determinant portion. ### Claim 2 (depends on 1) The component according to claim 1, comprising a primer set including: the first forward primer; the second forward primer; the probe having a sequence complementary to a reporter binding portion; and a reverse primer having a sequence complementary to a complementary region on the target nucleic acid. ### Claim 3 (depends on 2) The component of claim 2, wherein the complementary region on the target nucleic acid is about 50-200 bp downstream of a complementary region between the first forward primer and the target sequence. ### Claim 4 (depends on 2) The component of claim 2, wherein the first forward primer and the second forward primer have different Tm values. ### Claim 5 (depends on 2) The component claim 2, wherein the mismatch portion has a length of 1-15 bases. ### Claim 6 (depends on 2) The component of claim 2, wherein a distance between the amplification determinant portion and the mismatch portion is about 0-20 bases. ### Claim 7 (depends on 2) The component of claim 2, wherein the upstream detection portion is configured not to sequentially match or complement with the target sequence, or to hybridize with the target nucleic acid under a stringent condition. ### Claim 8 (depends on 2) The component of claim 2, wherein variation of the allele sequence is a point mutation. ### Claim 9 (depends on 2) A method for detecting a sequence variation of a target nucleic acid in a sample using digital PCR with the component of claim 2, wherein said sample is diluted to a limit and distributed randomly to 770-10,000,000 fractions, each fraction containing the primer set for simultaneous amplification across all fractions in a uniform thermal cycle, said method comprising: maintaining a reaction condition to allow the primer set to form pre-amplification complexes; initiating amplification reactions; detecting signals emitted from the probes in each fraction; and determining a quantitation for the target nucleic acid in the sample based on the signals detected. ### Claim 10 (depends on 9) The method of claim 9, wherein the complementary region between the reverse primer and the target nucleic acid is around 50-200 bp downstream of a complementary region between the first forward primer and the target sequence. ### Claim 11 (depends on 9) The method of claim 9, wherein a length of the mismatch portion is about 1-15 bases. ### Claim 12 (depends on 9) The method of claim 9, wherein a distance between the amplification determinant site portion and the mismatch portion is about 0-20 bases. ### Claim 13 (depends on 9) The method of claim 9, wherein the upstream detection portion of the first forward primer is configured not to sequentially match or complement with the target sequence, or to hybridize with the target nucleic acid under a stringent condition. ### Claim 14 (depends on 9) The method of claim 9, wherein said maintaining is performed at a different temperature from the said initiating. ### Claim 15 (depends on 9) The method of claim 9, wherein the first forward primer and the second forward primer are configured to have a different Tm value. ### Claim 16 (depends on 9) The method of claim 9, wherein said maintaining further comprising maintaining reaction conditions to allow 3-10 thermal cycles for the pre-amplification complexes to form. ### Claim 17 (depends on 9) The method of claim 9, wherein a variation of the sequence variation of the allele is a point mutation. ### Claim 18 (depends on 9) The method of claim 9, wherein the primer set includes multiple species of first forward primers and multiple species of probes. ### Claim 19 A composition, comprising: a molecular complex formed by a target nucleic acid encoding a target sequence, a first forward primer, a second forward primer, and a probe in complex with each other, wherein: said first forward primer comprising: a target sequence binding portion; and a detection portion upstream of the target sequence binding portion, and wherein: said detection portion comprises a second forward primer binding portion encoding a sequence complementary to the second forward primer located upstream to a reporter binding portion encoding a sequence complementary to the probe; and said target sequence binding portion comprises: a mismatch portion having a sequence not complementary to the target nucleic acid, and an amplification determinant portion, different from, and downstream to, the mismatch portion, the amplification determinant portion having a sequence complementary to a gene allele or a variant thereof encoded by the target nucleic acid; wherein there are no other bases downstream of the amplification determinant portion. ### Claim 20 (depends on 2) A kit comprising the component of claim 2, and configured to perform digital PCR to detect at least one allele of a sequence variation of a target nucleic acid, the kit comprising: at least one primer set, wherein said target sequence binding portion encodes a sequence complementary to the sequence of the allele or a variation thereof.