# US 11,299,775 B2 · Method for designing primer, primer, primer set, DNA amplification method, and analysis method

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- Number: US 11,299,775 B2
- Document ID: US11299775B2
- Status: Granted
- Kind: B2
- Application number: 15/921,061
- Filed: 2018-03-14
- Published: 2018-07-19
- Granted: 2022-04-12
- Earliest claimed: 2015-09-30
- Assignees: FUJIFILM Corporation
- Applicants: FUJIFILM Corporation
- Inventors: Takayuki Tsujimoto
- Web page: https://patentgrep.com/patent/US11299775B2

## CPC

- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6853: using modified primers or templates
- C12N 15/09: Recombinant DNA-technology
- C12Q 1/68: involving nucleic acids
- G16B 25/00: ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B 25/20: Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
- 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)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

A method for designing a primer supplied to a PCR reaction includes a primer designing step of defining a 5′-end-side base sequence of the primer so as to include a base sequence or its complementary base sequence, the base sequence enabling identification of, among thermal cycles in the PCR reaction, a synthesis cycle of a DNA molecule in an amplification product obtained by the PCR reaction performed with the primer, the identification being based on base-sequence data obtained by sequencing the amplification product.

## Claims

### Claim 1

A method for designing a primer supplied to a PCR reaction, the method comprising:
a primer designing step of including a base sequence or its complementary base sequence on a 5′-end-side base sequence of the primer, the base sequence enabling identification at which cycle of thermal cycles in the PCR reaction a DNA molecule in an amplification product obtained by the PCR reaction performed with the primer is synthesized, the identification being based on base-sequence data obtained by sequencing the amplification product.

### Claim 2 (depends on 1)

The method for designing a primer supplied to a PCR reaction according to claim 1,
wherein, in the primer designing step,
the primer is a primer used in N-th and N+1-th thermal cycles,
N represents an odd number of 1 or more,
when N=1, the step includes coupling a non-complementary DNA fragment to a 5′ end of a complementary DNA fragment,
the complementary DNA fragment being constituted by a base sequence that complementarily binds to a 3′ end side of an amplification target region in amplification target DNA; the non-complementary DNA fragment being constituted by a base sequence that does not complementarily bind to the amplification target region; and
when N≥3, the step includes coupling a non-complementary DNA fragment to a 5′ end of a complementary DNA fragment,
the complementary DNA fragment being constituted by a base sequence of a non-complementary DNA fragment of a primer used in an N−2-th thermal cycle; the non-complementary DNA fragment being constituted by a base sequence that is different from each one of base sequences of non-complementary DNA fragments of primers used in 1st to N−2-th thermal cycles, and that does not complementarily bind to the amplification target region.

### Claim 3 (depends on 1)

The method for designing a primer supplied to a PCR reaction according to claim 1,
wherein, in the primer designing step,
the primer is a primer used in an N-th thermal cycle,
N represents an integer of 1 or more,
when N=1, the step includes coupling a non-complementary DNA fragment to a 5′ end of a complementary DNA fragment,
the complementary DNA fragment being constituted by a base sequence that complementarily binds to a 3′ end side of an amplification target region in amplification target DNA; the non-complementary DNA fragment being constituted by a base sequence that does not complementarily bind to the amplification target region;
when N=2, the step includes coupling a non-complementary DNA fragment to a 5′ end of a complementary DNA fragment,
the complementary DNA fragment being constituted by a base sequence that complementarily binds to a 3′ end side of an amplification target region in amplification target DNA; the non-complementary DNA fragment being constituted by a base sequence that is different from the base sequence of the non-complementary DNA fragment of the primer used in the 1st thermal cycle, and that does not complementarily bind to the amplification target region; and
when N≥3, the step includes coupling a non-complementary DNA fragment to a 5′ end of a complementary DNA fragment,
the complementary DNA fragment being constituted by a base sequence of a non-complementary DNA fragment of a primer used in an N−2-th thermal cycle; the non-complementary DNA fragment being constituted by a base sequence that is different from each one of base sequences of non-complementary DNA fragments of primers used in 1st to N−1-th thermal cycles, and that does not complementarily bind to the amplification target region.

### Claim 4 (depends on 1)

A primer designed by the method for designing a primer according to claim 1.

### Claim 5 (depends on 4)

A primer set supplied to a PCR reaction, the primer set comprising the primer according to claim 4.

### Claim 6 (depends on 5)

A primer set supplied to a PCR reaction, the primer set comprising a plurality of the primer sets according to claim 5.

### Claim 7 (depends on 5)

A DNA amplification method comprising supplying the primer set according to claim 5 to a PCR reaction.

### Claim 8 (depends on 5)

A method for analyzing base-sequence data of an amplification product obtained by supplying the primer set according to claim 5 to a PCR reaction to amplify a plurality of regions, the method comprising:
identifying, in the PCR reaction, the synthesis cycles of DNA molecules in amplification products corresponding to regions,
determining the numbers of successful PCR times for the regions, and
performing correction between coverages of the regions in accordance with the numbers to ensure uniform PCR amplification efficiency between the regions.

### Claim 9 (depends on 6)

A method for analyzing base-sequence data of an amplification product obtained by supplying the primer set according to claim 6 to a PCR reaction to amplify a plurality of regions, the method comprising:
identifying, in the PCR reaction, the synthesis cycles of DNA molecules in amplification products corresponding to regions,
determining the numbers of successful PCR times for the regions, and
performing correction between coverages of the regions in accordance with the numbers to ensure uniform PCR amplification efficiency between the regions.

### Claim 10 (depends on 5)

A method for analyzing base-sequence data of an amplification product obtained by supplying the primer set according to claim 5 to a PCR reaction to amplify a region including at least one locus, the method comprising:
identifying, in the PCR reaction, the synthesis cycles of DNA molecules in amplification products corresponding to alleles,
determining the numbers of successful PCR times for the alleles, and
performing correction between coverages of the alleles in accordance with the numbers to ensure uniform PCR amplification efficiency between the alleles.

### Claim 11 (depends on 6)

A method for analyzing base-sequence data of an amplification product obtained by supplying the primer set according to claim 6 to a PCR reaction to amplify a region including at least one locus, the method comprising:
identifying, in the PCR reaction, the synthesis cycles of DNA molecules in amplification products corresponding to alleles,
determining the numbers of successful PCR times for the alleles, and
performing correction between coverages of the alleles in accordance with the numbers to ensure uniform PCR amplification efficiency between the alleles.