# US 10,738,354 B2 · Targeted enrichment of long nucleotide sequences using microfluidic partitioning

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- Number: US 10,738,354 B2
- Document ID: US10738354B2
- Status: Granted
- Kind: B2
- Application number: 15/739,541
- Filed: 2016-06-24
- Published: 2018-06-28
- Granted: 2020-08-11
- Earliest claimed: 2015-06-26
- Assignees: Samplix Aps
- Applicants: Samplix ApS
- Inventors: Marie Just Mikkelsen; Thomas Kvist; Esben Bjørn Madsen
- Web page: https://patentgrep.com/patent/US10738354B2

## CPC

- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/686: Polymerase chain reaction [PCR]
- 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

The invention relates to a method for enrichment or isolation of a long nucleotide fragment comprising a known nucleotide sequence element, i.e. a sequence encoding a conserved active site or domain, the method being applicable i.a. to high throughput screening for DNA fragments containing a known sequence element. The methods include, inter alia, steps of providing a liquid sample of mixed DNA molecules comprising one or more specific target DNA molecules, formation of a multiple of liquid droplets, specific detection of droplets containing at least one of the target DNA molecules, physically selecting droplets containing at least one of the target DNA molecules, and general amplification of the DNA molecules in the coalesced selected droplets.

## Claims

### Claim 1

An in vitro method for enriching one or more target DNA molecules from a sample of mixed DNA molecules comprising the steps of:
a) providing a liquid sample of mixed DNA molecules comprising one or more specific target DNA molecule and reagents for specific detection of at least one of said target DNA molecules,
b) formation of a multiple of liquid droplets from said liquid sample,
c) specific detection of droplets containing at least one of said target DNA molecules, wherein each droplet contains less than 0.5 of said one of more target DNA molecule on average, and
d) physically selecting droplets containing at least one of said target DNA molecules,
e) general amplification of DNA molecules in the selected droplets from step d), where the DNA molecules are in a total amount of less than 300 fg, wherein the selected droplets are coalesced and general amplification reagents are added to said coalesced selected droplets to form a general amplification reaction mixture, and wherein water-in-oil droplets are formed from the reaction mixture.

### Claim 2

An in vitro method for enriching for one or more target DNA molecule from a sample of mixed DNA molecules comprising the steps of:
a) providing a liquid sample of mixed DNA molecules comprising one or more specific target DNA molecule and reagents for specific detection of at least one of said target DNA molecules,
b) formation of a multiple of liquid droplets each comprising mixed DNA molecules from said liquid sample,
c) specific detection of droplets containing at least one of said target DNA molecules, wherein each droplet contains less than 0.5 of said one of more target DNA molecule on average, and
d) physically selecting droplets containing at least one of said target DNA molecules, wherein the frequency of the target DNA molecule compared to its frequency in the sample of mixed DNA molecules in step (a) is increased between 0.01×(total number of droplets containing DNA molecules)×(number of droplets with target DNA)−1 and 100×(total number of droplets containing DNA molecules)×(number of droplets with target DNA)−1,
e) general amplification of the mixed DNA molecules in the selected droplets from step d), where the mixed DNA molecules are in a total amount of less than 300 fg, wherein the selected droplets are coalesced and general amplification reagents are added to said coalesced selected droplets to form a general amplification reaction mixture, and wherein water-in-oil droplets are formed from the reaction mixture.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the total number of droplets in step b) is at least 5×105.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the reagents are PCR reagents and specific detection of said one or more target DNA molecule is performed by PCR.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said target DNA molecule comprises one or more unique consecutive sequence of at least 40 nucleotides.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the general amplification of DNA in step (e) is performed by Multiple Displacement Amplification.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the reagents for specific detection contain dUTP.

### Claim 8 (depends on 7)

The method according to claim 7, wherein step (d) further comprises the step of inactivating, degrading or removing DNA produced for specific detection of said one or more target DNA molecule.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the step of inactivating is performed using uracil-DNA N-glycosylase.

### Claim 10 (depends on 1)

The method according to claim 1, further comprising a step (f) of repeating steps (a) to (e), wherein the DNA molecules in said liquid sample of repeated step (a) are derived from the general amplification products generated from the DNA molecules in the coalesced droplets selected in step (e).

### Claim 11 (depends on 1)

The method according to claim 1, wherein each of the droplets formed in step b) have an average diameter from 1 to 100 μm.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the droplets formed in step b) contain on average at least 10−7 target DNA molecule per droplet.

### Claim 13 (depends on 1)

The method according to claim 1, wherein each of said one of more target DNA molecule of step a) comprises 1,000 to 108 nucleic acid base pairs.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the general amplification product generated in step e) comprises one of more amplified target DNA molecule, wherein each amplified target DNA molecule comprises 500 to 150,000 nucleic acid base pairs.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the target DNA molecule is derived from the genome of a cell.

### Claim 16 (depends on 1)

The method according to claim 1, wherein the water-in-oil droplets formed in step e) have a volume of 1 pL to 1 nL.

### Claim 17 (depends on 1)

The method according to claim 1, wherein in step c), each droplet contains less than 0.1 of said one of more target DNA molecule on average.

### Claim 18 (depends on 12)

The method according to claim 12, wherein the droplets formed in step b) contain on average from 10−4 to 10−5 target DNA molecule per droplet.

### Claim 19 (depends on 13)

The method according to claim 13, wherein each of said one of more target DNA molecule of step a) comprises 10,000 to 100,000 nucleic acid base pairs.

### Claim 20 (depends on 14)

The method according to claim 14, wherein the general amplification product generated in step e) comprises one of more amplified target DNA molecule, wherein each amplified target DNA molecule comprises 5,000 to 50,000 nucleic acid base pairs.