# US 11,078,477 B2 · Automated isolation and chemical reaction(s) of nucleic acids

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- Number: US 11,078,477 B2
- Document ID: US11078477B2
- Status: Granted
- Kind: B2
- Application number: 16/663,212
- Filed: 2019-10-24
- Published: 2020-04-16
- Granted: 2021-08-03
- Earliest claimed: 2014-10-17
- Assignees: Biochain Institute, Inc.
- Applicants: Biochain Institute, Inc.
- Inventors: Xiaoliang Han; James Jianming Wang; Tong Lu
- Web page: https://patentgrep.com/patent/US11078477B2

## CPC

- C12N 15/1013: {by using magnetic beads}
- B01L 3/50851: {specially adapted for heating or cooling samples}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 1/10: in the liquid or fluent state {(burettes, pipettes B01L3/02; sampling of ground water E02D1/06; metering by volume of fluids or fluent solid material G01F11/00, G01F13/00)}
- B01L 2300/0858: Side walls
- G01N 35/0098: {involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation (magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C)}
- G01N 2035/00564: {Handling or washing solid phase elements, e.g. beads}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- 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

The present teachings relate to methods, kits and devices for performing automated sequential nucleic acid isolation and conversion/purification in a single closed system. In various embodiments, the present teaching enable a user to (i) load a device with test samples, reagents and consumables; (ii) select or program the device for the desired nucleic acid isolation and subsequent chemical treatment and/or conversion reaction(s) without further user intervention; and recovering the isolated and treated and/or converted nucleic acid at the conclusion of the program once the device is activated.

## Claims

### Claim 1

A method for performing sequential nucleic acid isolation and conversion/purification reaction(s) in a closed automated device which includes:
(A) a plurality of reaction reservoirs;
(B) a heating unit configured to heat the plurality of reaction reservoirs;
(C) a magnet configured to be elevated and lowered relative to the reaction reservoirs;
the method comprising:
a. contacting the nucleic acid-containing sample with an extraction reagent in a first reservoir of the plurality of the reaction chambers to thereby extract the nucleic acid from the sample;
b. binding the extracted nucleic acid with a first plurality of magnetic beads in the first reservoir;
c. while holding the magnetic beads down by elevating the magnet relative to the first plurality of magnetic beads, removing liquid supernatant formed in the first reservoir;
d. eluting the bound extracted nucleic acid from the first plurality of magnetic beads using a first elution buffer while subjecting the first reservoir to a first elevated temperature by the heating unit for a first predetermined duration of time to thereby obtain an isolated nucleic acid eluent;
e. while holding the magnetic beads down by elevating the magnet relative to the first plurality of magnetic beads, transferring the isolated nucleic acid eluent to a second reservoir of the plurality of reaction reservoirs, the second reaction reservoir containing a conversion reagent;
f. reacting said isolated nucleic acid with the conversion reagent in the second reservoir while subjecting the second reservoir to a second elevated temperature by the heating unit for a second predetermined duration of time, to thereby obtain a modified nucleic acid;
g. binding the modified nucleic acid with a second plurality of magnetic beads;
h. while holding the magnetic beads down by elevating the magnet relative to the second plurality of magnetic beads, removing liquid supernatant formed;
j. eluting the bound modified nucleic acid from the second plurality of magnetic beads using a second elution buffer or a second series of elution buffers to thereby obtain a purified modified nucleic acid eluent.

### Claim 2 (depends on 1)

The method of claim 1, wherein said conversion is methylation.

### Claim 3 (depends on 2)

The method of claim 2, wherein the methylation comprises bisulfite conversion.

### Claim 4 (depends on 3)

The method of claim 3, wherein the bisulfite conversion is a deamination of cytosine into uracil of said isolated nucleic acid.

### Claim 5 (depends on 3)

The method of claim 3, wherein the conversion reagent comprises bisulfite.

### Claim 6 (depends on 3)

The method of claim 3, further comprising a step of desulfonation.

### Claim 7 (depends on 1)

The method of claim 1, wherein the sample is selected from the group consisting of whole blood, plasma, serum, buffy coat, saliva, cheek swab, sputum, stool, urine, cerebral spinal fluid, a cell, a tissue and a formalin fixed paraffin embedded (FFPE) sample.

### Claim 8 (depends on 1)

The method of claim 1, wherein the nucleic acid is DNA, RNA, cDNA or a combination thereof.