# US 11,305,282 B2 · Devices, processes, and systems for determination of nucleic acid sequence, expression, copy number, or methylation changes using combined nuclease, ligase, polymerase, and sequencing reactions

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- Number: US 11,305,282 B2
- Document ID: US11305282B2
- Status: Granted
- Kind: B2
- Application number: 16/498,893
- Filed: 2018-03-29
- Published: 2020-02-06
- Granted: 2022-04-19
- Earliest claimed: 2017-03-29
- Assignees: Cornell University
- Applicants: Cornell University
- Inventors: Francis Barany
- Web page: https://patentgrep.com/patent/US11305282B2

## CPC

- B01L 3/502761: {specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules}
- B01L 3/50851: {specially adapted for heating or cooling samples}
- B01F 33/3017: {Mixing chamber}
- B01F 33/813: {mixing simultaneously in two or more mixing receptacles}
- C12Q 1/6869: Methods for sequencing
- B01L 3/527: {for a plurality of reagents}
- B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples}
- B01L 2200/027: for microfluidic devices
- B01L 2200/0605: Metering of fluids
- B01L 2200/0642: Filling fluids into wells by specific techniques
- B01L 2200/0668: Trapping microscopic beads
- B01L 2200/0673: Handling of plugs of fluid surrounded by immiscible fluid
- B01L 2200/16: Reagents, handling or storing thereof
- B01L 2300/0681: Filter
- B01L 2300/0819: Microarrays; Biochips
- B01L 2300/0829: Multi-well plates; Microtitration plates
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- B01L 2300/087: Multiple sequential chambers
- B01L 2300/161: Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
- B01L 2300/165: Specific details about hydrophobic, oleophobic surfaces
- B01L 2300/1805: Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L 2400/0622: distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- 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 present invention relates to methods, devices, instruments, processes, and systems for the highly specific, targeted molecular analysis of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, IncRNA, circulating tumor cells, or total blood cells. The technology enables highly sensitive identification and enumeration of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using spatial multiplexing and combined nuclease, ligation, polymerase, and sequencing reactions. Such technology may be used for non-invasive early detection of cancer, non-invasive cancer prognosis, and monitoring both treatment efficacy and disease recurrence of cancer.

## Claims

### Claim 1

A system for identifying a plurality of nucleic acid molecules in a sample, said system comprising:
(i) an inlet port and
(ii) a cartridge defining a space containing:
multiple primary reaction chambers fluidically coupled to said inlet port to receive material from said inlet port and produce primary reaction chamber products from the material and
a product capture housing enclosing a solid support with a plurality of separate columns of a plurality of product capture subdivisions configured in separate rows and columns, with each separate product capture subdivision comprising an array of a plurality of individual hydrophilic micro-pores or micro-wells, wherein each of said individual hydrophilic micro-pores or micro-wells is separated by hydrophobic surfaces where primary reaction products are further reacted to create array products which are detected in the micro-pores or micro-wells, wherein one or more of the columns of separate product capture subdivisions receive material which has passed through one of said multiple primary reaction chambers.

### Claim 2 (depends on 1)

The system of claim 1 further comprising:
(iii) an outlet port for discharging material from said product capture housing.

### Claim 3 (depends on 1)

The system of claim 1, wherein the space defined by said cartridge further comprises:
one or more initial reaction chambers into which said inlet port discharges material and from which material is discharged into said multiple primary reaction chambers.

### Claim 4 (depends on 1)

The system of claim 1, wherein the space defined by the cartridge further comprises:
multiple secondary reaction chambers, one or more of which are fluidically coupled to one of said multiple primary reaction chambers to receive material from one of said multiple primary reaction chambers and
multiple mixing chambers each fluidically coupled to one of said multiple secondary reaction chambers to receive material from one of said multiple secondary reaction chambers and to discharge material to said product capture housing so that each column of separate product capture subdivisions is fluidically coupled to one of said multiple mixing chambers to receive material from one of said multiple mixing chambers.

### Claim 5 (depends on 4)

The system of claim 4, wherein at least some of said multiple primary and secondary reaction chambers are configured to maintain a trough of liquid in said multiple primary and secondary reaction chambers.

### Claim 6 (depends on 4)

The system of claim 4, wherein said multiple primary and secondary reaction chambers each have an internal baffle to maintain a trough of liquid in said multiple primary and secondary reaction chambers.

### Claim 7 (depends on 4)

The system of claim 4, wherein said multiple primary and/or secondary reaction chambers each have one or more of internal baffles to maintain a plurality of troughs of liquid in said multiple primary and secondary reaction chambers.

### Claim 8 (depends on 4)

The system of claim 4, wherein each of said mixing chambers include a divider extending from a position proximate to where material enters said mixing chamber to a position proximate to where material leaves said mixing chambers.

### Claim 9 (depends on 4)

The system of claim 4, wherein each of said mixing chambers include a first surface which is highly hydrophobic and a second surface spaced from, and less hydrophobic than, the first surface, wherein the first and second surfaces extend from a position proximate to where material enters each of said mixing chambers to a position proximate to where material leaves each of said mixing chambers.

### Claim 10 (depends on 4)

The system of claim 4, wherein said primary reaction chambers and/or said secondary reaction chambers comprise an internal surface on to which oligonucleotide primers or probes can be spotted.

### Claim 11 (depends on 1)

The system of claim 1, wherein the product capture subdivisions comprise the array of the plurality of individual micro-pores each having opposed first and second open ends with the first end having a large diameter and the second end having a diameter which is smaller than that of the first end.

### Claim 12 (depends on 1)

The system of claim 1, wherein the product capture subdivisions comprise the array of the plurality of individual micro-wells each having an open end and a closed end.

### Claim 13 (depends on 4)

The system of claim 4, wherein said product capture housing comprises:
a plurality of fluid channels to permit material to pass from said multiple mixing chambers, through a column of the product capture subdivisions into contact with the array of micro-pores or micro-wells in those subdivisions.

### Claim 14 (depends on 1)

The system of claim 1 further comprising:
one or more valves for selectively introducing or removing reagents or reactants into or out of the cartridge through said inlet.

### Claim 15 (depends on 2)

The system of claim 2 further comprising:
one or more valves for selectively introducing or removing reagents or reactants into or out of said product capture housing through said outlet port and/or through a location in said product capture housing distal from said outlet port.

### Claim 16 (depends on 1)

The system of claim 1 further comprising:
one or more heating elements in said cartridge in a position proximate to said primary reaction chamber and/or in a position proximate to said product capture housing.

### Claim 17 (depends on 3)

The system of claim 3 further comprising:
one or more heating elements in said cartridge in a position proximate to said initial reaction chambers.

### Claim 18 (depends on 4)

The system of claim 4 further comprising:
one or more heating elements in said cartridge in a position proximate to one of said secondary reaction chambers and/or in a position proximate to one of said multiple mixing chambers.

### Claim 19 (depends on 1)

A method for preparing a system for identifying a plurality of nucleic acid molecules in a sample, said method comprising:
providing the system of claim 1 and applying universal tag or capture oligonucleotide primers or probes to the micro-pores or micro-wells of the product capture subdivisions on the solid support within said product capture housing, whereby the universal tag or capture oligonucleotide primers or probes are retained within the micro-pores or micro-wells.

### Claim 20 (depends on 19)

The method of claim 19, further comprising:
filling the one or more primary reaction chambers with primary reaction oligonucleotide probes or primers each having a first portion comprising a nucleotide sequence complementary to a portion of target nucleic acids in the sample.

### Claim 21 (depends on 20)

A process of identifying a plurality of nucleic acid molecules in a sample using the system prepared by the method of claim 20, wherein the primary reaction oligonucleotide probes or primers further comprise a second portion comprising a nucleotide sequence the same or complementary to a portion of the universal tag or capture oligonucleotide primers, retained within the micro-pores or micro-wells and wherein, following said filling the one or more primary reaction chambers and optionally said filling the one or more secondary reaction chambers, if present, said process further comprising:
conducting the primary and/or secondary reactions in said system and
detecting the presence of target nucleic acid molecules in the sample in the micro-wells or micro-pores after said conducting the primary and/or secondary reactions in said system.

### Claim 22 (depends on 11)

The system of claim 11 further comprising:
a mesh screen covering the second ends of the micro-pores in said product capture housing.

### Claim 23 (depends on 11)

The system of claim 11 further comprising:
a bead placed in the individual micro-pores.

### Claim 24 (depends on 13)

The system of claim 13, wherein said plurality of fluid channels are located above and below the solid support.

### Claim 25 (depends on 13)

The system of claim 13, wherein said plurality of fluid channels are located above the solid support.