# US 11,898,200 B2 · Method for detecting single strand breaks in DNA

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- Number: US 11,898,200 B2
- Document ID: US11898200B2
- Status: Granted
- Kind: B2
- Application number: 17/014,322
- Filed: 2020-09-08
- Published: 2021-03-11
- Granted: 2024-02-13
- Earliest claimed: 2019-09-05
- Assignees: Huaqiao University
- Applicants: Huaqiao University
- Inventors: Philipp Kapranov; Huifen Cao; Lorena Salazar-García; Fan Gao; Dongyang Xu; Ye Cai; Xueer Han; Fang Wang; Lu Tang
- Web page: https://patentgrep.com/patent/US11898200B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6869: Methods for sequencing
- 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 disclosure discloses a method for detecting single strand breaks (SSBs) in DNA based on the following steps. First, DNA of interest is fragmented with a method that generates 3′ ends that cannot be tailed. Second, the available 3′ ends of the fragmented DNA corresponding to the pre-existing breaks are tailed. Third, SSBs are captured and their positions are identified genome-wide based on the following steps: (1) the tailed fragments are linearly amplified using a chimeric 5′-DNA-RNA-3′ primer; (2) the products of primer extension are tailed at the 3′ ends; (3) the desired products are amplified by PCR with oligonucleotides containing Illumina® adaptor sequences complementary to both tails and subjected to next-generation sequencing (NGS); 4) finally, positions of SSBs are revealed through the analysis of sequencing results.

## Claims

### Claim 1

A method for detecting single strand breaks (SSBs) in deoxyribonucleic acid (DNA) comprising:
fragmenting DNA with the SSBs to obtain fragmented DNA with available 3′ ends of the SSBs using a method that generates unavailable 3′ ends that cannot be tailed to generate fragmented DNA;
adding a first tail to the available 3′ ends of the SSBs of the fragmented DNA to generate tailed fragments; and
capturing and identifying positions of the SSBs genome-wide, wherein the capturing and identifying comprises:
linearly amplifying the tailed fragments using cycles of primer extension with a chimeric 5′-DNA-RNA-3′ primer;
adding a second tail to 3′ ends of products of the primer extension to obtain desired products;
amplifying the desired products by a polymerase chain reaction (PCR) with oligonucleotides comprising adaptor sequences complementary to both the first tail and the second tail and subjecting to next-generation sequencing (NGS) to generate sequencing results; and
revealing the positions of the SSBs through analysis of the sequencing results of the NGS.

### Claim 2 (depends on 1)

The method for detecting SSBs in DNA of claim 1, wherein:
the fragmenting comprises:
fragmenting the DNA with a micrococcal nuclease (MNase) that generates 3′-phosphate ends.

### Claim 3 (depends on 1)

The method for detecting SSBs in DNA of claim 1, wherein the fragmenting comprises fragmenting the DNA to a range of 150 to 500 base pairs.

### Claim 4 (depends on 1)

The method for detecting SSBs in DNA of claim 1, wherein bases of the first tail and bases of the second tail are not complementary.

### Claim 5 (depends on 1)

The method for detecting SSBs in DNA of claim 1, wherein the chimeric 5′-DNA-RNA-3′ primer is annealed to the first tail.

### Claim 6 (depends on 1)

The method for detecting SSBs in DNA of claim 1, wherein a length of dCTP used in tailing the second tail is 10 to 100 bases.

### Claim 7 (depends on 1)

The method for detecting SSBs in DNA of claim 1, wherein the adaptor sequences are adaptor sequences applicable to second and higher generation sequencing systems.

### Claim 8 (depends on 1)

The method for detecting SSBs in DNA of claim 1, wherein revealing the positions of the SSBs comprises:
(1) selecting paired-end reads, where a first read starts with a corresponding number of complementary bases of the second tail and a second read starts with a corresponding number of complementary bases of the first tail,
(2) aligning the paired-end reads after bases corresponding to PCR primers have been removed to assemblies of specified genomes, wherein only pairs of reads where the first read and the second read uniquely mapped to a genome of the specified genomes with preset configuration and spacing are kept, and
(3) removing pairs of reads where a fraction of T's in a 20-base sequence of 5′ upstream of an alignment of the second read of the paired-end reads is >40%, and defining an SSB as a first base in the second read of remaining read pairs of the paired-end reads.

### Claim 9 (depends on 1)

The method for detecting SSBs in DNA of claim 1, wherein an SSB of the SSBs is present in a first read, or in a second read of a read pair or in a single read.

### Claim 10 (depends on 8)

The method for detecting SSBs in DNA of claim 8, wherein defining the SSB comprises defining the SSB as the first base after 12 T's in the second read.