阅读说明：本技术 一种dna可逆保护和分离的方法 (Method for reversible protection and separation of DNA ) 是由 曹博 张清华 刘莉莉 季红 于 2020-06-11 设计创作，主要内容包括：本发明提供了一种对DNA进行可逆保护和分离的方法,首先将目标DNA分子的5’端进行磷酸化；然后将5’端腺苷化修饰；终止反应后获得的样品中加入对腺苷化修饰DNA敏感的核酸外切酶消化模板；最后将获得的腺苷化修饰DNA,即分离所得目标DNA,进行测序鉴定等技术分析,所得序列的5’端即为腺苷化修饰位点。本发明提供的方法,填补了现有技术无法精确定位基因组DNA上断裂位点的空白,能够实现对不同长度和不同来源的DNA样品进行断裂位点的定量和定位分析,使用简便,易于操作,而且对样品无特殊要求,准确率高、检测背景影响低、分辨率高。(The invention provides a method for reversible protection and separation of DNA, firstly phosphorylating the 5' end of a target DNA molecule; then the 5' end is modified by adenylation; adding an exonuclease digestion template sensitive to the adenylation modified DNA into a sample obtained after terminating the reaction; and finally, carrying out technical analysis such as sequencing identification on the obtained adenylation modified DNA, namely the target DNA obtained by separation, and obtaining a sequence with the 5' end as the adenylation modified site. The method provided by the invention fills the blank that the prior art can not accurately position the fracture site on the genome DNA, can realize the quantitative and positioning analysis of the fracture site on DNA samples with different lengths and different sources, is simple and convenient to use and easy to operate, has no special requirements on the samples, and has high accuracy, low detection background influence and high resolution.)

1. A method for reversible protection and isolation of DNA, comprising the steps of:

(1) subjecting the DNA molecules to an enzymatic treatment to obtain a sample containing 5' phosphorylated DNA;

(2) unwinding the sample containing the 5' phosphorylated DNA to obtain single-stranded DNA;

(3) carrying out 5 'adenylation labeling on the single-stranded DNA to obtain a sample containing 5' adenylation DNA;

(4) digesting the sample obtained in the step (3) by using adenylation sensitive 5 ' → 3 ' exonuclease, removing single-stranded DNA which is not modified by 5 ' adenylation, and purifying to obtain an adenylation modified target DNA molecule;

(5) and (3) carrying out de-adenylation treatment on the adenylation modified target DNA molecule to obtain the target DNA molecule.

2. The method according to claim 1, wherein in step (1), the DNA molecule is double-stranded or single-stranded; when the DNA molecule is single-stranded, the step (2) is omitted.

3. The method according to claim 1, wherein in step (1), the enzyme is an enzyme capable of converting a DNA having a 5 'hydroxyl group into a 5' phosphorylated DNA; the enzyme is selected from T4 polynucleotide kinase or excision repair enzyme involved in DNA damage site;

in step (4), the adenylation sensitive 5 '→ 3' exonuclease is selected from one or a combination of T5 exonuclease or RecJ exonuclease.

4. The method of claim 1, wherein in step (2), the unwinding process is thermal denaturation.

5. The method of claim 1, wherein step (1) (3) (5) further comprises a purification process for the reacted sample.

6. A method for detecting sites of damage and modification to DNA molecules using the method of any of claims 1 to 5, comprising the steps of:

(i) extracting DNA molecules, crushing, dephosphorizing and obtaining a sample;

(ii) obtaining a target DNA molecule according to the method;

(iii) and sequencing, analyzing and comparing the target DNA molecules to obtain the damaged or modified sites.

7. The method of claim 6, wherein in step (i), the step of disrupting is ultrasonication; the size of the disruption is preferably 200-500 bp.

8. The method of claim 6, wherein in step (iii), the sequencing is selected from Sanger sequencing or Illumina sequencing.

9. The method of claim 8, wherein in step (iii), the sequencing step is Illumina sequencing, and further comprises the following steps: and converting the target-removed DNA molecules into double-stranded DNA, and performing PCR amplification to obtain a product for Illumina sequencing.

Technical Field

The invention belongs to the field of molecular biology and biomedicine, and particularly relates to a method for reversibly protecting and separating DNA macromolecules.

Background

DNA molecules store the genetic information on which organisms live and multiply, and therefore maintaining the integrity of DNA is critical to cells. However, various factors in the external environment and inside the organism cause damage or alteration of DNA molecules, such as ultraviolet rays, radiation, carcinogenic chemicals, oxidative stress generated during the metabolism of the cell itself, and the like. These lesions disrupt the integrity of the genome and threaten the stability of the genome. It is now widely recognized that DNA damage is a major cause of cancer and many other age-related diseases, and is therefore a very important problem for human health.

Among the many types of DNA damage, Strand breaks (Strand break) are the most recognized type of damage to cells because they block DNA replication, transcription, etc., and may also lead to recombination events. Therefore, DNA strand breakage is a research hotspot in the field of life sciences. Among them, the study of the location and the regularity of the occurrence of strand breaks in genomic DNA is the basis for understanding such lesions.

Currently, there are two main techniques for locating DNA break sites across the entire genome:BaranelloDSB-Seq, SSB-Seq techniques andPhilipp Kapranovthe SSiNGLe technology of the subject group. The former uses biotin and digoxin to label the double-stranded break site and single-stranded break site, respectively, and then affinity enriches the labeled DNA fragments and combines with second-generation sequencing for analysis. The SSiNGLe technique uses micrococcal nuclease (MNase) to digest DNA during DNA fragmentation to generate 3 'phosphate ends, followed by terminal transferase (TDT) to add PolyA tails to label capture DNA fragmentation sites with 3' hydroxyl ends, and to combine with next-generation sequencing. Both methods, while allowing genome-wide localization of DNA break sites, have limitations in their application. For example, DSB-and SSB-seq have low resolution for the localization of the cleavage site, high background during sequencing; while the SSiNGLe technique does not detect breaks occurring in DNA Adenine (Adenine, a).

Therefore, in view of the importance of DNA fragmentation and the limitations of current identification techniques, the present invention develops a way to locate DNA fragmentation sites with low cost, high sensitivity, high resolution by means of reversible protection and isolation of target DNA molecules, and further applies to the study of locating various types of damage and modification of DNA. The technology can greatly promote scientific research and clinical application in the fields of DNA damage-repair process, cancer generation mechanism and prevention, drug safety evaluation, gene therapy, genetic diseases and the like.

Disclosure of Invention

Aiming at the problems of high background, low resolution and low accuracy of the existing method for detecting DNA damage, the invention provides a reversible protection and separation method of DNA, which can be used for detecting DNA damage, can position DNA damage sites with high precision, can be applied to DNA samples with different lengths and different sources, and can separate and analyze target DNA at single molecule and single nucleotide levels.

In order to achieve the purpose, the invention adopts the following technical scheme.

A method for reversible protection and isolation of DNA comprising the steps of:

(1) subjecting the DNA molecules to an enzymatic treatment to obtain a sample containing 5' phosphorylated DNA;

(2) unwinding the sample containing the 5' phosphorylated DNA to obtain single-stranded DNA;

(3) carrying out 5 'adenylation labeling on the single-stranded DNA to obtain a sample containing 5' adenylation DNA;

(4) digesting the sample obtained in the step (3) by using adenylation sensitive 5 ' → 3 ' exonuclease, removing single-stranded DNA which is not modified by 5 ' adenylation, and purifying to obtain an adenylation modified target DNA molecule;

(5) and (3) carrying out de-adenylation treatment on the adenylation modified target DNA molecule to obtain the target DNA molecule.

In the step (1), the source of the DNA molecule is not limited, and the DNA molecule can be artificially synthesized or extracted from animals, plants or microorganisms; DNA extraction methods commonly used in the art, such as phenol extraction, isopropanol precipitation, CTAB method, etc., may be used depending on the sample, and a commercial kit may also be used.

In step (1), the DNA molecule may be double-stranded or single-stranded. Alternatively, when the DNA molecule is single-stranded, step (2) may be omitted.

In step (1), the enzyme is an enzyme capable of converting a DNA having a 5 'hydroxyl group into a 5' phosphorylated DNA, e.g., T4 polynucleotide kinase; or an excision repair enzyme involved in DNA damage sites, wherein 5' phosphate ends generated by the enzymes can be DNA glycosylase and endonuclease, and one or more of DNA glycosylase such as Uracil DNA Glycosylase (UDG), 8-hydroxy guanine DNA glycosidase (hOGGl), formamidopyrimidine DNA glycosylase (FPG), Thymine DNA Glycosylase (TDG) and the like and endonuclease IV are selected according to the fragments to be detected.

Preferably, in step (2), the unwinding process is thermal denaturation. Specifically, the steps for obtaining single-stranded DNA are: the DNA was heat-denatured and then rapidly placed on ice to maintain the single-stranded state.

In step (3), the 5 'adenylation marker is a marker for modifying the 5' end of the DNA by an enzyme. The enzyme is a commonly used DNA adenylate enzyme, e.g., Mth RNA Ligase, T4 DNA Ligase, etc. The DNA adenylating enzyme adenylates the 5' end of DNA in the presence of ATP. In the examples of the present invention, a reaction was carried out using a kit containing Mth RNA Ligase and ATP.

In step (4), the adenylation sensitive 5 '→ 3' exonuclease, including but not limited to T5 exonuclease, RecJ exonuclease and any adenylation sensitive exonuclease such as.

Preferably, steps (1), (3) and (5) further comprise a purification treatment of the reacted sample.

The above methods can be used to detect DNA molecule modification and damage.

A method for detecting DNA molecule damage and modification sites by adopting the method comprises the following steps:

(i) extracting DNA molecules, crushing, dephosphorizing and obtaining a sample;

(ii) obtaining a target DNA molecule according to the method;

(iii) and sequencing, analyzing and comparing the target DNA molecules to obtain the damaged or modified sites.

In step (i), the step of disruption is ultrasonication; the size of the disruption is preferably 200-500 bp.

In step (iii), the sequencing is selected from, but not limited to, Sanger sequencing, Illumina sequencing; samples containing a single site were Sanger sequenced and samples containing multiple sites were Illumina sequenced. Preferably, the sequencing of step (iii) by Illumina further comprises the following steps: and converting the target-removed DNA molecules into double-stranded DNA, and performing PCR amplification to obtain a product for Illumina sequencing.

The principle of the invention is as follows:

firstly, carrying out thermal denaturation on a DNA sample, and then carrying out 5' terminal phosphorylation treatment on a target DNA molecule; adenosine transferase is utilized to carry out adenylation reversible modification on the 5' end of the phosphorylated DNA, so that the target DNA molecule is protected; then digesting unprotected DNA in a sample by adenylation sensitive 5 '→ 3' exonuclease, preserving the 5 'end adenylation modified DNA against nuclease hydrolysis, and purifying the 5' end adenylation modified DNA after template digestion to eliminate background influence; then 5 'end adenylation modification is eliminated through a deaminase, so that the sample recovers 5' end phosphorylation modification, and reversible protection and separation of target DNA molecules are realized; sequencing the DNA with the 5 'end removed from the adenylation modification, wherein the 5' end of the sequence obtained by sequencing is the original protection site, namely the breaking site of the DNA.

The invention has the following advantages:

the method for capturing and separating the target DNA fragments provided by the invention realizes the quantitative and positioning analysis of the target DNA with different lengths and different sources by utilizing the reversible protection of the 5' tail end of the DNA, is simple and convenient to use, is easy to operate, has no special requirements on samples, and has high accuracy; the detection background influence is low, the resolution ratio is high, and the method can be widely applied to the fields of molecular diagnosis, chemotherapy drug safety evaluation, cancer occurrence and prevention, molecular biology research, gene therapy and the like.

Drawings

FIG. 1 is a schematic diagram of a procedure for reversible protection and isolation of DNA;

FIG. 2 is a graph showing the result of adenylation modification of a short-chain DNA sample;

FIG. 3 is a graph showing the results of hydrolysis resistance of adenylated DNA;

FIG. 4 is a graph of the results of adenylated DNA polyadenylation;

FIG. 5 is a diagram showing the result of detection for precisely locating a damaged site of short-chain DNA;

FIG. 6 is a diagram showing the results of detection of precisely located genomic DNA damage sites.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.