# US 9,753,007 B1 · Isotachophoretic focusing of nucleic acids

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- Number: US 9,753,007 B1
- Document ID: US9753007B1
- Status: Granted
- Kind: B1
- Application number: 14/497,050
- Filed: 2014-09-25
- Granted: 2017-09-05
- Earliest claimed: 2009-03-03
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Robert D. Chambers; Juan G. Santiago; Alexandre Persat; Reto B. Schoch; Mostafa Ronaghi
- Web page: https://patentgrep.com/patent/US9753007B1

## CPC

- G01N 27/447: using electrophoresis
- B01D 57/02: by electrophoresis (treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26)
- C12N 15/101: {by chromatography, e.g. electrophoresis, ion-exchange, reverse phase}
- 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)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method and system are presented for fast and efficient isolation, purification and quantitation of nucleic acids from complex biological samples using isotachophoresis in microchannels. In an embodiment, a sieving medium may be used to enhance selectivity. In another embodiment, PCR-friendly chemistries are used to purify nucleic acids from complex biological samples and yield nucleic acids ready for further analysis including for PCR. In another embodiment, small RNAs from biological samples are extracted, isolated, preconcentrated and quantitated using on-chip ITP with a high efficiency sieving medium. The invention enables fast concentration and separation (takes 10s to 100s of seconds) of nucleic acids with high selectivity and using lower volumes of reagents (order of 10s of μL to focus less than 1 μg/μL of nucleic acid).

## Claims

### Claim 1

An isotachophoretic method for concentrating a target nucleic acid and for separating said target nucleic acid from at least one other macromolecule in a sample, said method comprising the steps of:
(a) applying a sample comprising a mixture of a target nucleic acid and ionic macromolecules that are not target nucleic acids to a sample well connected to at least one liquid channel;
(b) causing said sample comprising said mixture of a target nucleic acid and ionic macromolecules that are not target nucleic acids to be in contact with a trailing electrolyte (TE) and a leading electrolyte (LE) in said at least one liquid channel, said TE having an effective-mobility magnitude greater than at least one of said ionic macromolecules that are not target nucleic acids and less than said target nucleic acid, said LE having an effective-mobility magnitude greater than said target nucleic acid, and wherein said LE and TE contacting said sample are in free solution, and are at a pH of from 4 to 10; and
(c) applying a voltage difference along said at least one liquid channel, said at least one liquid channel containing said sample, said LE, and said TE, to cause concentration of said target nucleic acid in an isotachophoresis interface between LE and TE and separation of said at least one of said ionic macromolecules that are not target nucleic acids from said isotachophoresis interface between LE and TE.

### Claim 2 (depends on 1)

The method of claim 1 wherein said sample well and said at least one liquid channel are located within a microfluidic device.

### Claim 3 (depends on 1)

The method of claim 1 further comprising adding a polymer sieving agent to said at least one liquid channel to change an effective-mobility magnitude of one or both of said target nucleic acid and said at least one of said ionic macromolecules that are not target nucleic acids.

### Claim 4 (depends on 3)

The method of claim 3 wherein said sieving agent is selected from the group consisting of block copolymer, linear polymers, and cross-linked polymers.

### Claim 5 (depends on 1)

The method of claim 1 further comprising adding an agent for suppressing electroosmotic flow to said at least one liquid channel.

### Claim 6 (depends on 5)

The method of claim 5 wherein said agent for suppressing electroosmotic flow is selected from the group consisting of polylactams, substituted polyacrylamide derivatives, water soluble methylhydroxyethyl derivatives of cellulose, polyvinylalcohol, polyvinylpyrrolidones and polyethyleneglycols.

### Claim 7 (depends on 6)

The method of claim 6 wherein said polylactam is polyvinylpyrrolidone.

### Claim 8 (depends on 1)

The method of claim 1 wherein said at least one liquid channel divides at channel bifurcations to distribute various contents of said sample to various channels.

### Claim 9 (depends on 1)

The method of claim 1, wherein said sample is treated with a cell lysis agent prior to applying said sample to said sample well.

### Claim 10 (depends on 1)

The method of claim 1, wherein said sample is treated with a cell release agent to release said target nucleic acid from bound protein, if present, prior to applying said sample to said sample well.

### Claim 11 (depends on 10)

The method of claim 10 wherein said release agent is a protease.

### Claim 12 (depends on 1)

The method of claim 1 wherein said sample is mixed with said TE prior to entering said at least one liquid channel.

### Claim 13 (depends on 1)

The method of claim 1 further comprising removing concentrated target nucleic acid from an interface between said LE and said TE.

### Claim 14 (depends on 1)

The method of claim 1 wherein said target nucleic acid is genomic DNA.

### Claim 15 (depends on 1)

The method of claim 1, wherein said at least one liquid channel is a liquid microchannel.

### Claim 16 (depends on 1)

The method of claim 1, wherein said sample is mixed with said LE prior to entering said at least one liquid channel.

### Claim 17 (depends on 1)

The method of claim 1, wherein said at least one liquid channel is connected to a channel comprising said LE and a channel comprising said TE.

### Claim 18 (depends on 1)

The method of claim 1, wherein said sample contacts said LE and TE in said at least one liquid channel at the same time.

### Claim 19 (depends on 1)

The method of claim 1, wherein said sample contacts said TE prior to contacting said LE in said at least one liquid channel.

### Claim 20 (depends on 1)

The method of claim 1, wherein said sample contacts said LE prior to contacting said TE in said at least one liquid channel.

### Claim 21 (depends on 1)

The method of claim 1, wherein said sample is mixed with a spacer prior to entering said at least one liquid channel.

### Claim 22 (depends on 1)

The method of claim 1, wherein said target nucleic acid is RNA.

### Claim 23 (depends on 1)

The method of claim 1, wherein said target nucleic acid comprises DNA and RNA.

### Claim 24 (depends on 1)

The method of claim 1, wherein said at least one of said ionic macromolecules that are not target nucleic acids comprise protein.

### Claim 25 (depends on 1)

The method of claim 1, wherein said target nucleic acid comprises DNA and said ionic macromolecules that are not target nucleic acids comprise protein.

### Claim 26 (depends on 1)

The method of claim 1, wherein said method does not cause focusing of said at least one of said non-target ionic macromolecules in an isotachophoresis interface.

### Claim 27 (depends on 1)

The method of claim 1, wherein mobility of said LE and said TE is parallel to an electric field produced by said voltage difference.

### Claim 28 (depends on 1)

The method of claim 1, wherein said sample is selected from the group consisting of: whole blood, serum, and plasma.

### Claim 29 (depends on 28)

The method of claim 28, wherein said sample is a whole blood sample.

### Claim 30 (depends on 28)

The method of claim 28, wherein said sample is a plasma sample.

### Claim 31 (depends on 1)

The method of claim 1, wherein said sample comprises lysed cell membranes.

### Claim 32 (depends on 1)

The method of claim 1, wherein said sample comprises whole cells.

### Claim 33 (depends on 1)

The method of claim 1, wherein said sample further comprises buffer or water.

### Claim 34

An isotachophoretic method for concentrating small RNA from a sample comprising a mixture of macromolecules comprising small RNA, proteins, and longer RNA, comprising:
(a) applying a sample comprising a mixture of macromolecules comprising small RNA, proteins, and longer RNA to a sample well connected to at least one liquid channel;
(b) causing said sample comprising a mixture of macromolecules comprising small RNA, proteins, and longer RNA to be in contact with a trailing electrolyte (TE) and a leading electrolyte (LE) in said at least one liquid channel, said TE having an effective-mobility magnitude less than said small RNA and greater than said proteins in the sample, and said LE having an effective-mobility magnitude greater than said small RNA, wherein said LE and said TE are in free solution, and are at a pH which causes effective mobility of proteins to be different from that of the small RNA; and
(c) applying a voltage along said at least one liquid channel containing said sample, said LE, and said TE to cause concentration of said small RNA in an isotachophoresis interface between said LE and said TE, and to cause separation of said proteins and longer RNA from said isotachophoresis interface between said LE and said TE, wherein said LE and said TE are at a pH which causes effective mobility of proteins to be lower than that of said small RNA.

### Claim 35 (depends on 34)

The method of claim 34 further comprising the step of treating said sample with an RNAse inhibitor.

### Claim 36 (depends on 34)

The method of claim 34 further comprising adding a sieving agent to said LE.

### Claim 37 (depends on 36)

The method of claim 36 wherein said sieving agent is a block copolymer of ethylene oxide and propylene oxide.

### Claim 38 (depends on 34)

The method of claim 34 further comprising treating said sample with one or more of DNAse and protease.

### Claim 39 (depends on 38)

The method of claim 38 wherein said protease is Proteinase K.

### Claim 40 (depends on 34)

The method of claim 34 further comprising adding a protein denaturing agent to said at least one liquid channel.

### Claim 41 (depends on 34)

The method of claim 34 wherein said sample is mixed with said TE prior to entering said at least one liquid channel.