# US 2016/0355877 A1 · Methods of Detecting Target Nucleic Acids

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- Number: US 2016/0355877 A1
- Document ID: US20160355877A1
- Status: Published application
- Kind: A1
- Application number: 15/241,711
- Filed: 2016-08-19
- Published: 2016-12-08
- Earliest claimed: 2006-03-12
- Assignees: Applied Biosystems, LLC
- Applicants: Applied Biosystems LLC
- Inventors: Muhammad A. SHARAF; Timothy WOUDENBERG; Khairuzzaman Bashar MULLAH
- Web page: https://patentgrep.com/patent/US20160355877A1

## CPC

- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6823: Release of bound markers
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6832: Enhancement of hybridisation reaction
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- G01N 27/44726: {using specific dyes, markers or binding molecules}
- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- 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 disclosure relates to methods of identifying target nucleic acids by using coded molecules and its analysis by translocation through a nanopore. Generally, coded molecules are subject to a target polynucleotide dependent modification. The modified coded molecule is detected by isolating the modified coded molecules from the unmodified coded molecules prior to analysis through the nanopore or by detecting a change in the signal pattern of the coded molecule when analyzed through the nanopore.

## Claims

### Claim at position 1

1-73. (canceled)

### Claim 74

A method of detecting a plurality of target polynucleotides, the method comprising:
a) contacting a population of coded molecules with a plurality of target polynucleotides, wherein the population of coded molecules comprises a plurality of subpopulations and each coded molecule of each subpopulation comprises:
(i) one or more block polymer regions and a target probe capable of hybridizing to a target polynucleotide, wherein the target probe of each subpopulation hybridizes to a different target polynucleotide; and
(ii) a detectable signal pattern distinguishable amongst the plurality of subpopulations;
b) modifying coded molecules of the population of coded molecules by modifying the target probe with a modifying agent following hybridizing the target probe with the different target polynucleotide, wherein the modified target probe is indicative of the different target polynucleotide;
c) translocating the modified coded molecules through a nanopore and detecting the signal pattern; and
d) associating the detected signal pattern to a specific subpopulation of coded molecules in the plurality of subpopulations, wherein a difference in the detected signal pattern compared to the signal pattern of the specific subpopulation indicates the presence of the different target polynucleotide.

### Claim 75 (depends on 74)

The method of claim 74, wherein associating includes comparing the detected signal pattern to a signal pattern of the specific subpopulation.

### Claim 76 (depends on 74)

The method of claim 74, in which the target polynucleotide comprises a 5-prime region and a 3-prime region, and the target probe comprises a 3-prime terminal sequence that hybridizes to the 5-prime region of the target polynucleotide, and wherein the modifying agent is a template-dependent polymerase and the modification is extension of the hybridized 3-prime region of the target probe.

### Claim 77 (depends on 76)

The method of claim 76, in which the 3-prime terminal sequence of the target probe comprises a 3-prime terminal nucleotide that interrogates a site of nucleotide polymorphism on the target polynucleotide.

### Claim 78 (depends on 76)

The method of claim 76, in which the target polynucleotide comprises a circular nucleic acid, wherein the circular nucleic acid is a ligated open circle probe (OCP).

### Claim 79 (depends on 74)

The method of claim 74, in which the target polynucleotide comprises adjacent first and second regions, and the method further comprises hybridizing a ligation probe to the target polynucleotide, wherein the ligation probe hybridizes to the first region and the target probe hybridizes to the second region of the target polynucleotide such that a terminus of the ligation probe and a terminus of the target probe are adjacent, and wherein the modifying agent is a ligase and the modification is ligation of the ligation probe to the target probe.

### Claim 80 (depends on 79)

The method of claim 79, in which the terminus of the target probe comprises a terminal nucleotide that interrogates a site of nucleotide polymorphism on the target polynucleotide.

### Claim 81 (depends on 79)

The method of claim 79, in which the terminus of the ligation probe comprises a terminal nucleotide that interrogates a site of nucleotide polymorphism on the target polynucleotide.

### Claim 82 (depends on 74)

The method of claim 74, in which the target polynucleotide comprises adjacent first and second regions, and the target probe comprises a 5-prime region and a 3-prime region, and wherein the method further comprises hybridizing a FLAP probe to the target polynucleotide, wherein the FLAP probe comprises a 3-prime segment that hybridizes to the first region, and the 3-prime region of the target probe hybridizes to the second region such that the 3-prime segment of the FLAP probe and the 3-prime region of the target probe are adjacently hybridized to the target polynucleotide to form a FLAP substrate, and wherein the modifying agent is a FLAP endonuclease and the modification is cleavage of the target probe.

### Claim 83 (depends on 82)

The method of claim 82, in which the 5-prime region of the target probe is non-complementary to the target polynucleotide.

### Claim 84 (depends on 82)

The method of claim 82, in which the FLAP probe further comprises a 3-prime unpaired segment that overlaps with the 5-prime region of the target probe in the FLAP substrate, thereby forming a double FLAP substrate.

### Claim 85 (depends on 82)

The method of claim 82, in which the target probe interrogates a site of nucleotide polymorphism on the target polynucleotide.

### Claim 86 (depends on 82)

The method of claim 82, in which the FLAP probe interrogates a site of nucleotide polymorphism on the target polynucleotide.

### Claim 87 (depends on 74)

The method of claim 74, in which the hybridization of the target probe to the target polynucleotide forms an endonuclease recognition site and a corresponding endonuclease cleavage site, and wherein the modifying agent is an endonuclease that recognizes the recognition site and the modification is cleavage of the target probe.

### Claim 88 (depends on 87)

The method of claim 87, in which the endonuclease recognition site is a sequence-specific endonuclease recognition site and wherein the modifying agent is a sequence specific endonuclease active on the recognition site.

### Claim 89 (depends on 74)

The method of claim 74, in which the modifying agent is a double-stranded specific exonuclease suitable to act on the target probe hybridized to the target polynucleotide and the modification is degradation of all or a portion of the target probe.

### Claim 90 (depends on 74)

The method of claim 74, in which the coded molecule comprises a chimeric polymer.

### Claim 91 (depends on 90)

The method of claim 90, in which the chimeric polymer comprises nucleobase and non-nucleobase polymers.

### Claim 92 (depends on 74)

The method of claim 74, in which the coded molecule comprises at least two block polymers, and wherein the two block polymers are separated by a non-block polymer segment.

### Claim 93 (depends on 74)

The method of claim 74, in which the block polymer comprises dinucleotide repeats.