# US 2021/0340611 A1 · Digital polymerase chain reaction method for detecting nucleic acids in samples

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- Number: US 2021/0340611 A1
- Document ID: US20210340611A1
- Status: Published application
- Kind: A1
- Application number: 17/283,809
- Filed: 2019-10-09
- Published: 2021-11-04
- Earliest claimed: 2018-10-09
- Assignees: Academia Sinica
- Applicants: Academia Sinica
- Inventors: Kuo-Ping CHIU
- Web page: https://patentgrep.com/patent/US20210340611A1

## CPC

- C12Q 1/6855: Ligating adaptors
- G16B 25/20: Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
- C12Q 1/6853: using modified primers or templates
- G16B 40/10: Signal processing, e.g. from mass spectrometry [MS] or from 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
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

The present invention relates to a method for detecting nucleic acid (NA) molecules in samples. More particularly, the present invention relates to an improved digital PCR-based method for detecting specific nucleic acid sequence(s). The present invention is useful for research and diagnostic applications with increased sensitivity and accuracy. The present invention also provides a kit for performing the method for assessingnucleic acids in samples as described herein.

## Claims

### Claim 1

A method for analyzing nucleic acids in a sample, said sample containing one or more linear, double stranded nucleic acid fragment(s) (NA fragment(s)), said method comprising the steps of:
(a) subjecting the sample to a 3′-A tailing reaction allowing for adding an adenine nucleotide (A) to the 3′-tail of the NA fragment(s) to produce 3′-adenine nucleotide (3′-A) overhang nucleic acid fragment(s) (3′-A overhang NA fragment(s));
(b) providing a 3′-thymine (T) or 3′-uracil (U) nucleotide overhang double-stranded homogenous adapter which comprises a P oligo strand carrying a 5′-phosphate for ligation to the 3′-A overhang NA fragment(s) and a T/U oligo strand carrying a 3′-T or 3′-U without a 5′-phosphate, wherein the T/U oligo strand is complimentary to the P oligo strand except at the 3′-T or 3′-U of the T/U oligo strand;
(c) subjecting the sample of (a) to a ligation reaction allowing for ligating the homogenous adapter to the 3′-A overhang NA fragment(s) at both ends to produce adapter-ligated nucleic acid fragment(s) (adapter-ligated NA fragment(s));
(d) combining the sample of (c) with polymerase chain reaction (PCR) reagents and detection reagents to provide amplification/detection-ready sample, wherein the PCR reagents include a single-type primer having the nucleic acid sequence of the T/U oligo strand for amplification and the detection reagents include one or more fluorescent probe(s) producing fluorescent signals and specifically hybridizing with the NA fragment(s) for detection;
(e) partitioning the amplification/detection-ready sample of (d) into a plurality of partitions, each containing limited copy of the adapter-ligated NA fragment(s);
(f) performing PCR in each partition using the adapter-ligated NA fragment(s) as template(s) and the single-type primer as both forward and reverse primers to amplify the adapter-ligated NA fragment(s); and
(g) assessing the fluorescent signal(s) in each fraction.

### Claim 2 (depends on 1)

The method of claim 1, wherein step (g) includes detecting the fluorescent color (signal) for all partitions containing amplified adapter-ligated NA fragment(s) and counting the number of factions with expected fluorescent color and thus determining the estimated amount of the NA fragment(s) in the sample.

### Claim 3 (depends on 1)

The method of claim 1, wherein in step (e), more than 50% of the partitions containing no more than one copy of the adapter-ligated NA fragment(s).

### Claim 4 (depends on 1)

The method of claim 1, wherein in step (e), each partition contains at least one copy of the adapter-ligated NA fragment(s).

### Claim 5 (depends on 1)

The method of claim 1, wherein the NA fragment(s) comprises a nucleic acid sequence indicative of a healthy/diseased state of the subject.

### Claim 6 (depends on 1)

The method of claim 1, wherein the homogenous adapter of step (b) does not self-ligate.

### Claim 7 (depends on 1)

The method of claim 1, wherein the homogenous adapter of step (b) has 3′-T or 3′-U overhang in the T/U oligo strand and 3′-non-A overhang in the P oligo strand.

### Claim 8 (depends on 1)

The method of claim 1, wherein the homogenous adapter of step (b) has one end which is 3′-T overhang and the other end which is a blunt end.

### Claim 9 (depends on 1)

The method of claim 1, wherein the sample is obtained from a body fluid sample.

### Claim 10 (depends on 1)

The method of claim 1, wherein the NA fragment(s) in the sample are cell-free DNAs.

### Claim 11 (depends on 1)

The method of claim 1, wherein prior to step (a) further comprising performing an end-repair reaction to the NA fragment(s).

### Claim 12 (depends on 1)

The method of claim 1, wherein the PCR of step (0 is performed by oil emulsion or droplet PCR, or well-based PCR.

### Claim 13 (depends on 1)

The method of claim 1, wherein the assaying of step (g) is performed by flow cytometry using fluorescent probes.

### Claim 14 (depends on 1)

The method of claim 1, further comprising subjecting the sample prior to step (a) to a reverse transcription-PCR (RT-PCR) to convert the RNAs to linear, double-stranded complementary DNA (cDNA).

### Claim 15

A kit for performing a method for measuring the quantity of nucleic acid fragment(s) in a sample, comprising
(i) adapter ligation reagents comprising a homogenous adapter, a ligation buffer and a ligase, wherein the adapter comprises a P oligo strand carrying a 5′-phosphate and a T/U oligo strand carrying a 3′-T or 3-U without a 5′-phosphate, wherein the T/U oligo strand is complimentary to the P oligo strand except at the 3′-T or 3′-U of the T/U oligo strand, wherein the homogenous adapter is capable of ligating to the nucleic acid fragment(s) at both ends, wherein the nucleic acid fragment(s) has a 3′-A overhang;
(ii) PCR reagents comprising a single-type primer as the sole primer having the nucleic acid sequence of the T/U oligo strand, dNTPs, a PCR buffer, and a DNA polymerase; and
(iii) detection reagents comprising one or more detectable probes having complementary sequences specifically hybridizing with the nucleic acid fragment(s).

### Claim 16 (depends on 15)

The kit of claim 15, further comprises instructions for use, wherein the instructions for use comprise instructions for performing a method comprising the steps of:
(a) subjecting the sample to a 3′-A tailing reaction allowing for adding an adenine nucleotide (A) to the 3′-tail of the NA fragment(s) to produce 3′-adenine nucleotide (3′-A) overhang nucleic acid fragment(s) (3′-A overhang NA fragment(s));
(b) providing a 3′-thymine (T) or 3′-uracil (U) nucleotide overhang double-stranded homogenous adapter which comprises a P oligo strand carrying a 5′-phosphate for ligation to the 3′-A overhang NA fragment(s) and a T/U oligo strand carrying a 3′-T or 3′-U without a 5′-phosphate, wherein the T/U oligo strand is complimentary to the P oligo strand except at the 3′-T or 3′-U of the T/U oligo strand;
(c) subjecting the sample of (a) to a ligation reaction allowing for ligating the homogenous adapter to the 3′-A overhang NA fragment(s) at both ends to produce adapter-ligated nucleic acid fragment(s) (adapter-ligated NA fragment(s));
(d) combining the sample of (c) with polymerase chain reaction (PCR) reagents and detection reagents to provide amplification/detection-ready sample, wherein the PCR reagents include a single-type primer having the nucleic acid sequence of the T/U oligo strand for amplification and the detection reagents include one or more fluorescent probe(s) producing fluorescent signals and specifically hybridizing with the NA fragment(s) for detection;
(e) partitioning the amplification/detection-ready sample of (d) into a plurality of partitions, each containing limited copy of the adapter-ligated NA fragment(s);
(f) performing PCR in each partition using the adapter-ligated NA fragment(s) as template(s) and the single-type primer as both forward and reverse primers to amplify the adapter-ligated NA fragment(s); and
(g) assessing the fluorescent signal(s) in each fraction.