# US 2020/0399677 A1 · Methods for differentially quantifying nucleic acids

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- Number: US 2020/0399677 A1
- Document ID: US20200399677A1
- Status: Published application
- Kind: A1
- Application number: 16/911,268
- Filed: 2020-06-24
- Published: 2020-12-24
- Earliest claimed: 2019-06-24
- Assignees: ChromaCode, Inc.
- Applicants: ChromaCode, Inc.
- Inventors: Christopher MacDonald; Lucien Jacky; Aditya Rajagopal
- Web page: https://patentgrep.com/patent/US20200399677A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/683: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
- C12Q 1/682: Signal amplification
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/76: Chemiluminescence; Bioluminescence
- C12Q 2600/154: Methylation markers
- C12Q 2600/156: Polymorphic or mutational markers
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Methods for differentially quantifying nucleic acids in a biological sample using differentially methylated loci include forming a digested biological sample in a solution of a methylation-sensitive restriction enzyme to cleave an unmethylated group of nucleic acids, forming a reaction mixture, amplifying the reaction mixture to generate a first signal from the a group of detection probes and a second signal from a second group of detection probes, determining a ratio comprising a first value derived from the first signal to a second value derived from the second signal, and identifying the proportion of nucleic acid in the biological sample originating from the first plurality of nucleic acids.

## Claims

### Claim 1

A method for differentially quantifying nucleic acids in a biological sample using differentially methylated loci, the method comprising:
providing a solution comprising a methylation-sensitive restriction enzyme;
providing a biological sample, wherein the biological sample comprises a first plurality of nucleic acids and a second plurality of nucleic acids and wherein each of the first and second pluralities of nucleic acids further comprise a group of methylated nucleic acids and a group of unmethylated nucleic acids;
forming a digested biological sample by combining the biological sample and the solution, such that the methylation-sensitive restriction enzyme cleaves the unmethylated group of nucleic acids;
forming a reaction mixture by combining the digested biological sample with:
a set of amplification oligomers configured to amplify the first and second pluralities of nucleic acids;
a first group of detection probes configured to anneal to a region of the first plurality of nucleic acids; and
a second group of detection probes configured to anneal to a region of the second plurality of nucleic acids;
amplifying the reaction mixture to generate a first signal from the first group of detection probes and a second signal from the second group of detection probes;
determining a ratio comprising a first value derived from the first signal to a second value derived from the second signal; and
identifying the proportion of nucleic acid in the biological sample originating from the first plurality of nucleic acids.

### Claim 2 (depends on 1)

The method of claim 1, further comprising partitioning the reaction mixture into reaction volumes prior to amplification.

### Claim 3 (depends on 2)

The method of claim 2, wherein the first and second signals each comprise cumulative signal measurements generated from a total number of amplified reaction volumes containing an amplicon from each of the first and second pluralities of nucleic acids.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first signal and the second signal are each generated in a single fluorescence channel.

### Claim 5 (depends on 1)

The method of claim 1, wherein the first signal and the second signal are generated in more than one fluorescence channel.

### Claim 6 (depends on 1)

The method of claim 1, wherein the reaction mixture further comprises one or more of buffers, reagents, a thermostable polymerase, and deoxyribonucleotide triphosphates.

### Claim 7 (depends on 1)

The method of claim 1, wherein the set of amplification oligomers comprises a forward amplification oligomer and a reverse amplification oligomer.

### Claim 8 (depends on 1)

The method of claim 1, wherein the biological sample comprises genomic DNA from one or more organism.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first group of detection probes and the second group of detection probes each further comprise a detectable label.

### Claim 10 (depends on 9)

The method of claim 9, wherein the detectable label is selected from the group consisting of a chemiluminescent label, a fluorescent label, or any combination thereof.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first group of detection probes and the second group of detection probes each further comprise a quencher.

### Claim 12 (depends on 1)

The method of claim 1, wherein the ratio is determined without quantifying the first and second plurality of nucleic acids.

### Claim 13 (depends on 1)

The method of claim 1, wherein identifying the proportion of nucleic acid in the biological sample originating from the first plurality of nucleic acids further comprises determining a fraction of methylated nucleic acids in the biological sample.

### Claim 14 (depends on 13)

The method of claim 13, wherein determining a fraction of methylated nucleic acids in the biological sample comprises comparing the ratio to a reference value.

### Claim 15 (depends on 1)

The method of claim 1, wherein the biological sample comprises whole blood from a pregnant female.

### Claim 16 (depends on 15)

The method of claim 15, wherein the whole blood comprises maternal nucleic acids and fetal nucleic acids.

### Claim 17 (depends on 1)

The method of claim 1, wherein the first plurality of nucleic acids comprises fetal nucleic acid sequences.

### Claim 18 (depends on 1)

The method of claim 1, wherein the second plurality of nucleic acids comprises maternal nucleic acid sequences.

### Claim 19 (depends on 1)

The method of claim 1, wherein the first group of detection probes are configured to detect a region of nucleic acid associated with fetal aneuploidy.

### Claim 20 (depends on 19)

The method of claim 19, wherein the region comprises a region of chromosome 22, chromosome 21, chromosome 18, chromosome 13, chromosome 9, chromosome 8, Y chromosome, or X chromosome.

### Claim 21 (depends on 1)

The method of claim 1, wherein the methylation-sensitive restriction enzyme comprises one or more of HpaII, AatII, AccII, Aor13HI, Aor51HI, BspT104I, BssHII, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HhaI, MluI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI and SnaBI.

### Claim 22

A kit for differentially quantifying nucleic acids in a biological sample using differentially methylated loci, the kit comprising:
a methylation-sensitive restriction enzyme;
a set of amplification oligomers configured to amplify a first plurality of nucleic acids and a second plurality of nucleic acids;
a first group of detection probes configured to anneal to the first plurality of nucleic acids; and
a second group of detection probes configured to anneal to the second plurality of nucleic acids;
wherein the set of amplification oligomers comprises a forward amplification oligomer and a reverse amplification oligomer; and
wherein the first group of detection probes and the second group of detection probes each contain a detectable label.

### Claim 23 (depends on 22)

The kit of claim 22, wherein the detectable label is selected from the group consisting of a chemiluminescent label, a fluorescent label, or any combination thereof.

### Claim 24 (depends on 22)

The kit of claim 22, wherein the first group of detection probes and the second group of detection probes each further comprise a quencher.

### Claim 25 (depends on 22)

The kit of claim 22, further comprising one or more of buffers, reagents, a thermostable polymerase, and deoxyribonucleotide triphosphates.

### Claim 26

A kit for differentially quantifying a fetal fraction in a biological sample using differentially methylated loci, the kit comprising:
a methylation sensitive-restriction enzyme;
a set of amplification oligomers configured to amplify a plurality of fetal nucleic acids and a plurality of maternal nucleic acids;
a first group of detection probes configured to anneal to the plurality of fetal nucleic acids;
a second group of detection probes configured to anneal to the plurality of maternal nucleic acids;
wherein the set of amplification oligomers comprises a forward amplification oligomer and a reverse amplification oligomer; and
wherein the first group of detection probes and the second group of detection probes each comprise a detectable label.

### Claim 27 (depends on 26)

The kit of claim 26, wherein the detectable label is selected from the group consisting of a chemiluminescent label, a fluorescent label, or any combination thereof.

### Claim 28 (depends on 26)

The kit of claim 26, wherein the first group of detection probes and the second group of detection probes each further comprise a quencher.

### Claim 29 (depends on 26)

The kit of claim 26, further comprising one or more of buffers, reagents, a thermostable polymerase, and deoxyribonucleotide triphosphates.