# US 11,898,201 B2 · Split-cycle and tape amplification

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

- Number: US 11,898,201 B2
- Document ID: US11898201B2
- Status: Granted
- Kind: B2
- Application number: 17/148,440
- Filed: 2021-01-13
- Published: 2021-07-08
- Granted: 2024-02-13
- Earliest claimed: 2015-12-30
- Assignees: Bio-Rad Laboratories, Inc.
- Applicants: Bio-Rad Laboratories, Inc.
- Inventors: Nicholas Heredia; Dianna Maar
- Web page: https://patentgrep.com/patent/US11898201B2

## CPC

- C12Q 1/6865: Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6851: Quantitative amplification
- C12Q 1/6853: using modified primers or templates
- C12Q 1/6858: Allele-specific amplification
- 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

Methods and compositions are provided for improved nucleic acid amplification assays. In some embodiments, the nucleic acid amplification assay is a tagged amplicon primer extension (TAPE) nucleic acid amplification reaction.

## Claims

### Claim 1

A plurality of mixture partitions, the individual mixture partitions comprising:
i) a mutation-specific 5′-tailed primer pair, wherein the mutation-specific 5′-tailed primer pair hybridizes to and specifically amplifies target DNA template molecules from a nucleic acid sample that comprise a mutant target sequence, if present, wherein the primers of the mutation-specific 5′-tailed primer pair comprise:
a) a 3′ hybridization region that specifically hybridizes to the mutant target sequence; and
b) a mutation-specific 5′ tail region that does not hybridize to any nucleic acid fragments in the nucleic acid sample;
ii) a wild-type-specific 5′-tailed primer pair, wherein the wild-type-specific 5′-tailed primer pair hybridizes to and specifically amplifies target DNA template molecules comprising a wild-type target sequence, if present, wherein the primers of the wild-type-specific 5′-tailed primer pair comprise:
a) a 3′ hybridization region that specifically hybridizes to the wild-type target sequence; and
b) a wild-type-specific 5′ tail region that does not hybridize to any nucleic acid fragments in the nucleic acid sample, wherein the wild-type-specific 5′ tail region is a different sequence than the mutation-specific 5′ tail region; and
iii) a mutation-specific flanking primer pair, wherein the mutation-specific flanking primer pair hybridizes to and specifically amplifies amplicons comprising the 5′ tail regions of the mutation-specific 5′-tailed primer pair, if present;
iv) a wild-type-specific flanking primer pair, wherein the wild-type-specific flanking primer pair hybridizes to and specifically amplifies amplicons comprising the 5′ tail regions of the wild-type-specific 5′-tailed primer pair, if present; and
v) a thermostable polymerase, wherein
at least some of the mixture partitions of the plurality of mixture partitions contain a target DNA template molecule that comprises a wild-type or mutant target sequence; and
at least some of the mixture partitions of the plurality of mixture partitions do not contain the target DNA template molecule.

### Claim 2 (depends on 1)

The plurality of mixture partitions of claim 1, wherein the mixture partitions comprise emulsion droplets.

### Claim 3 (depends on 1)

The plurality of mixture partitions of claim 1, wherein at least about 10% and no more than about 90% of the plurality of mixture partitions comprise multiple target DNA template molecules.

### Claim 4 (depends on 1)

The plurality of mixture partitions of claim 1 wherein the target DNA template in the mixture partitions is at an average concentration of about 1-5 copies per partition.

### Claim 5 (depends on 1)

The plurality of mixture partitions of claim 1, wherein at least one of the target DNA templates in the mixture partitions comprises a mutant target sequence.

### Claim 6 (depends on 1)

The plurality of mixture partitions of claim 1, wherein the plurality comprises from about 500 to about 10,000,000 mixture partitions.

### Claim 7 (depends on 1)

The plurality of mixture partitions of claim 1, wherein the 3′ hybridization regions of the wild-type and mutant 5′-tailed primer pairs comprise annealing temperatures to the wild-type and mutant target DNA template molecules respectively that are at least about 5° C. less than the annealing temperatures of the mutation-specific and wild-type-specific flanking primers.

### Claim 8 (depends on 1)

The plurality of mixture partitions of claim 1, wherein the 3′ hybridization regions of the wild-type and mutant 5′-tailed primer pairs comprise annealing temperatures to the wild-type and mutant target DNA template molecules respectively that are at least about 5° C. more than the annealing temperatures of the mutation-specific and wild-type-specific flanking primers.

### Claim 9 (depends on 1)

A method for quantitating a frequency of wild-type and mutant target nucleic acid fragments in a nucleic acid sample, the method comprising:
(a) forming a plurality of mixture partitions of claim 1;
(b) incubating the mixture partitions under thermal cycling conditions suitable for amplification of the target DNA template molecules by a polymerase chain reaction, wherein the thermal cycling conditions comprise a first set of temperature cycles and a second set of temperature cycles, wherein the second set of temperature cycles comprises an annealing temperature that is at least 5° C. higher or lower than an annealing temperature of the first set of temperature cycles;
(c) detecting the presence or absence of amplified target DNA template in the mixture partitions, wherein the detecting comprises, determining:
i) a number of wild-type mixture partitions comprising amplified target DNA template consisting of wild-type target sequence;
ii) a number of mutant mixture partitions comprising amplified target DNA template consisting of mutant target sequence; and
iii) a number of double-positive mixture partitions comprising amplified target DNA comprising mutant and wild-type target sequence; and
(d) determining from the number of wild-type, mutant, and double-positive mixture partitions the frequency of wild-type and mutant target nucleic acid fragments in the nucleic acid sample.