CN113832221A

CN113832221A - High-flux detection method of R ring

Info

Publication number: CN113832221A
Application number: CN202111076403.0A
Authority: CN
Inventors: 宋东亮; 王嫚; 侯策; 江翱; 陈晶晶; 刘倩; 卢瑶; 曹振
Original assignee: Yisheng Biotechnology Shanghai Co ltd
Current assignee: Yisheng Biotechnology Shanghai Co ltd
Priority date: 2021-09-14
Filing date: 2021-09-14
Publication date: 2021-12-24

Abstract

The invention provides a high-throughput detection method of an R ring, which comprises the steps of firstly using ddNTP to seal free 3' -OH of DNA in a sample; then converting RNA in the sample into DNA with biotin modification, and then performing DNA fragmentation and end repair; connecting a joint; enriching DNA modified by biotin by using streptavidin magnetic beads; strand-specific library amplification and sequencing. The invention provides a simple, convenient and rapid technology for detecting the R loop in a genome range, which can efficiently and accurately identify the R loop, has the advantages of simple operation of the whole process, short time consumption (not more than 4 h), wide applicability, high sensitivity and high accuracy, and is suitable for development and application of an R loop detection commodity kit.

Description

High-flux detection method of R ring

Technical Field

The invention relates to a simple and rapid high-flux detection method for an R ring, and belongs to the technical field of biology.

Background

The R-loop (R-loop) refers to a specific DNA-RNA structure on chromatin, consisting mainly of RNA: the ternary complex of double-stranded DNA and free single-stranded complementary DNA. DNA methylation and R loops are two important states on chromosomal DNA, have important regulation and control functions on the loose degree and accessibility of chromatin, the interaction between chromatin and the expression level of adjacent genes, and are widely involved in various physiological and pathological processes such as cell differentiation, embryonic development, individual aging, cytopathic effect and the like. Therefore, the R ring is an important marker for detecting the gene expression level and has an important role in the detection process of diseases such as tumors and the like.

However, no high-throughput sequencing product for simply and rapidly detecting the R loop exists in the market at present. The existing method can not be developed into an effective commercial product because of complex operation, strict requirements on materials and high false positive. Currently available R-ring detection techniques include the following:

(1) DRIP-seq series: the detection method mainly relies on the co-immunoprecipitation of DNA fragments by an antibody S9.6 which specifically recognizes the DNA-RNA hybrid chain. The method has the defects of low sensitivity, complex operation, long time (2 days), low specificity and accuracy and the like.

(2) The R-ring detection technology based on the ChIP-seq technology comprises the following steps: such as RChIP, relying on exogenous over-expressed RNaseH mutants to co-immunoprecipitate DNA/RNA regions of the chromatin and to library. The method can only carry out experiments on a cell level, and has the disadvantages of complicated operation, long time consumption (3 days) and low sensitivity.

(3) The R ring detection technology based on the CUT & Tag or CUT & RUN technology: such as MapR and Loop CUT & Tag, which rely on S9.6 antibodies or activity-deficient RNase H to localize the R Loop, on transposase or endonuclease to fragment and label the DNA at the R Loop, and then to library. The sensitivity of the method is higher than that of RChIP, but the method still has the problems of long time consumption (2 days), low sensitivity, poor specificity, complex operation and the like. In addition, the technology only can use normal living cells as starting materials, and the requirement on samples is high.

In summary, the existing R-loop detection technology mainly uses co-immunoprecipitation or an immunolabeling method for detection, which is tedious in operation, long in time consumption, high in requirements for sample types, and poor in specificity and accuracy, and greatly limits the popularization and application of the R-loop detection technology.

Disclosure of Invention

The invention aims to provide a simple and rapid high-throughput detection method for an R ring.

The technical scheme adopted by the invention is as follows: a high-throughput detection method of R ring is characterized by comprising the following steps:

(1) extracting DNA in a sample, and blocking free 3' -OH of the DNA in the sample by using ddNTP (ddNTP), wherein the blocking can prevent a false positive signal caused by the fact that a fracture region is extended and inserted into a base with biotin on the DNA;

(2) converting RNA in a sample into DNA with biotin modification by using ribonuclease and DNA polymerase, utilizing the characteristic of RNase H for specifically cutting RNA on a DNA-RNA double strand to generate a notch on the RNA of R-loop, then using the DNA polymerase to extend by taking biotin-labeled dUTP as a raw material, converting an RNA chain into a DNA chain with the biotin-labeled dUTP, and finally converting the DNA chain into double-stranded DNA with the biotin-dUTP on one chain;

(3) DNA fragmentation and end repair;

(4) connecting a joint;

(5) enriching DNA modified by biotin by streptavidin magnetic beads;

(6) strand-specific library amplification and sequencing.

Preferably, step (2) uses Escherichia coli DNA polymerase I and ribonuclease H.

Preferably, biotin-labeled dUTP is used in the amplification of RNA in the sample in step (2).

Preferably, the biotin-labeled dUTP is biotin-epsilon-aminocaproyl- [5- { 3-aminoallyl } -2 '-deoxyuridine-5' -triphosphate (biotin-11-dUTP) or 5- (N- [ N-biotin-epsilon-aminopropyl-gamma-aminobutyl ] -3-aminoacyl) -2 '-deoxyguanosine' -triphosphate (biotin-16-dUTP).

Preferably, the reaction system of step (2) is:

components	Dosage of
		Sample DNA treated in step (1)	20 ul
10 mM dA/G/CTP mix	0.2-1 ul
		10 mM biotin-11-dUTP or biotin-16-dUTP	0.2-1 ul
10 U/ul E. coli DNA Polymerase I	0.5-2 ul
		5 U/ul RNase H	0.1-2 ul
10× NEB Buffer™ 2	5 ul
		10 mM ATP mix	1 ul
Supplying DEPC water to	50 ul

Preferably, the reaction process of the step (2) is a reaction at 16 ℃ for 20-60 min.

Preferably, step (1) is specifically to add DNA polymerase and ddNTP to the sample DNA to block free 3' -OH of the sample DNA.

Preferably, the reaction system of step (1) is:

components	Dosage of
		Genomic DNA	1-5000 ng
10 mM ddNTP mix	0.1-1 ul
		10× NEB Buffer™ 2	2 ul
10 U/ul E. coli DNA Polymerase I	0.5-2 ul
		Supplying DEPC water to	20 ul

Preferably, the reaction process of step (2) is a reaction at 37 ℃ for 10-30 min, and DNA is recovered after the reaction is finished.

Preferably, the enrichment time in step (5) is 10-30 min.

Preferably, in step (6), the library amplification is performed using a dUTP-sensitive DNA polymerase.

The invention has the beneficial effects that:

the invention provides a simple, convenient and quick technology for detecting R ring in a genome range, which can efficiently and accurately identify the R ring and is named as DR Hybrid-seq. The process is as follows: blocking DNA free 3' OH with ddNTP; converting the RNA into DNA with biotin modification; DNA fragmentation and end repair; connecting a joint; enriching streptavidin magnetic beads; the strand-specific library was amplified and sequenced. The whole process is simple to operate, short in time consumption (not more than 4 h), wide in applicability, high in sensitivity and accuracy, and suitable for development and application of the R-ring detection commodity kit.

Drawings

FIG. 1 is a schematic diagram of DR Hybrid-seq principle.

FIG. 2 DR Hybrid-seq compares the advantages and disadvantages of other sequencing methods.

FIG. 3 Signal distribution of DR Hybrid-seq on genes.

FIG. 4 signal examples of DR Hybrid-seq at Myc and GAPDH genes.

Detailed Description

In order to further describe the concrete contents of the present invention, the present invention will be described in detail with reference to the following examples. The methods and reagents involved in the examples are well known to those skilled in the art, and it should be understood that the specific examples described below are merely illustrative of the invention and that the embodiments of the invention are not limited by the examples described below.

Example 1 DR Hybrid-seq procedure

(1) DNA extraction: genomic DNA of 24-well plate HEK293F cells was extracted using MolPure Cell/Tissue DNA Kit from the holy organisms next.

(2) Blocking of DNA free 3' OH with ddNTP

TABLE 1

Components	Dosage of
		Genomic DNA	1-5000 ng
10 mM ddNTP mix	0.1-1 ul
		10× NEB Buffer™ 2	2 ul
10 U/ul DNA Polymerase I (E. coli)	0.5-2 ul
		Supplying DEPC water to	20 ul

Reacting at 37 ℃ for 10-30 min. DEPC water was added to 50 ul. DNA was recovered using a DNA Clean & Concentrator ™ region-5 kit from Zymo Research, and eluted with 30 ul of DEPC water.

(3) Converting the RNA into DNA with biotin modification;

TABLE 2

Components	Dosage of
		DNA recovered by the above reaction	20 ul
10 mM dA/G/CTP mix	0.2-1 ul
		10 mM biotin-11-dUTP or biotin-16-dUTP	0.2-1 ul
10 U/ul DNA Polymerase I (E. coli)	0.5-2 ul
		5 U/ul RNase H	0.1-2 ul
10× NEB Buffer™ 2	5 ul
		10 mM ATP mix	1 ul
Supplying DEPC water to	50 ul

Reacting at 16 ℃ for 20-60 min. DNA was recovered using a DNA Clean & Concentrator ™ region-5 kit from Zymo Research, and eluted with 20 ul DEPC water.

(4) DNA fragmentation and end repair;

TABLE 4

Components	Dosage of
		DNA recovered by the above reaction	20 ul
Smearase® Mix	4 ul

Reacting at 30 ℃ for 10-20 min and at 72 ℃ for 20 min.

(5) Connecting a joint;

TABLE 5

Components	Dosage of
		DNA of the above reaction	24 ul
Ligation Enhancer	12
		Fast T4 DNA Ligase	2
DNA Adapter	2

The reaction was carried out at 20 ℃ for 15 min.

(6) Enriching streptavidin magnetic beads;

5ul of Dynabeads-M-270 streptavidin was taken, and the beads were washed 3 times with binding buffer and suspended in 10 ul of binding buffer. The beads were added to the ligation system and spun at room temperature for 15 minutes. The beads were washed 6 times with wash buffer at 50 ℃.

(7) Strand-specific library amplification and sequencing.

TABLE 6

Components	Dosage of
		2×Super Canace® II High-Fidelity Mix	25 ul
Primer mix	5
		H2O	20

The reaction solution prepared in Table 6 was added to the magnetic beads and the beads were resuspended. The reaction was carried out according to the procedure of table 7:

TABLE 7

After the reaction is finished, 45 ul Hieff NGS DNA Selection Beads are added, uniformly mixed by vortex or blowing, and incubated for 5 min at room temperature. The PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads and liquid, and after the solution cleared, the supernatant was carefully removed. The PCR tube was kept in the magnetic rack all the time, 200 ul of freshly prepared 80% ethanol was added to rinse the beads, and after incubation at room temperature for 30 s, the supernatant was carefully removed. The magnetic beads were washed repeatedly. Keeping the PCR tube in the magnetic frame all the time, opening the cover and drying in air for 2-3 min). 21 ul ddH was added₂And O, performing vortex oscillation or gently blowing and beating by using a pipette until the mixture is fully mixed, and standing for 5 min at room temperature. The PCR tube was centrifuged briefly and placed in a magnetic stand to stand, after the solution was clarified, 20 ul of the supernatant was carefully removed to a new PCR tube for library quantification, quality testing, and sequencing on the NovaSeq 6000 platform.

The DR Hybrid-seq principle diagram is shown in FIG. 1, and the whole DR Hybrid-seq flow takes no more than 4 h. FIG. 2 shows the comparison between the DR Hybrid-seq and other conventional high throughput sequencing technologies for detecting R-Loop, where the DR Hybrid-seq has the significant advantages of simple operation, short time consumption, wide application range, high sensitivity, high specificity and accuracy, etc. FIG. 3 shows the signal distribution of DR Hybrid-seq data on genes, similar to the results of other conventional high-throughput sequencing technologies for detecting R-Loop, wherein R-Loop is mainly distributed in the transcription promoter region of genes and a small amount is distributed in the transcription terminator region of genes (FIG. 4 is a specific example on Myc and GAPDH genes in the sequencing results), which also shows that R-Loop signals have obvious correlation with chromatin state and gene activity.

In conclusion, the invention provides a simple and rapid technology for detecting R loop in a genome range, which can efficiently and accurately identify the R loop and is named as DR Hybrid-seq. The process is as follows: blocking DNA free 3' OH with ddNTP; converting the RNA into DNA with biotin modification; DNA fragmentation and end repair; connecting a joint; enriching streptavidin magnetic beads; the strand-specific library was amplified and sequenced. The whole process is simple to operate, short in time consumption (not more than 4 h), wide in applicability, high in sensitivity and accuracy, and suitable for development and application of the R-ring detection commodity kit.

Claims

1. A high-throughput detection method of R ring is characterized by comprising the following steps:

(1) extracting DNA in a sample, and blocking free 3' -OH of the DNA in the sample by using ddNTP;

(2) converting RNA in the sample into DNA with biotin modification by using ribonuclease H and DNA polymerase;

(3) DNA fragmentation and end repair;

(4) connecting a joint;

(5) enriching DNA modified by biotin by streptavidin magnetic beads;

(6) strand-specific library amplification and sequencing.

2. The method for high-throughput detection of R-ring according to claim 1, wherein: in the step (2), Escherichia coli DNA polymerase I and ribonuclease H are used.

3. The high-throughput detection method for R-ring according to claim 1, characterized in that: biotin-labeled dUTP is used for amplification of RNA in the sample in step (2).

4. The high-throughput detection method for R ring according to claim 3, characterized in that: the biotin-labeled dUTP is biotin-11-dUTP or biotin-16-dUTP.

5. The high-throughput detection method for R ring according to claim 4, characterized in that: the reaction system of the step (2) is as follows:

。

6. The high-throughput detection method for R ring according to claim 5, characterized in that: the reaction process of the step (2) is reaction at 16 ℃ for 20-60 min.

7. The high-throughput detection method for R-ring according to claim 1, characterized in that: the step (1) is to add DNA polymerase and ddNTP into the sample DNA to block free 3' -OH of the sample DNA.

8. The high-throughput detection method for R-ring according to claim 7, characterized in that: the reaction system of the step (1) is as follows:

。

9. The high-throughput detection method for R-ring according to claim 8, characterized in that: the reaction process of the step (2) is reaction at 37 ℃ for 10-30 min, and DNA is recovered after the reaction is finished.

10. The high-throughput detection method for R-ring according to claim 1, characterized in that: the enrichment time in the step (5) is 10-30 min.

11. The high-throughput detection method for R-ring according to claim 1, characterized in that: in step (6), dUTP sensitive DNA polymerase is used for library amplification.