CN113717256B

CN113717256B - Fusion protein and application thereof

Info

Publication number: CN113717256B
Application number: CN202011308076.2A
Authority: CN
Inventors: 姚红杰; 李尧益; 王新秀; 黄赛南
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS; Bioisland Laboratory
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS; Bioisland Laboratory
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2023-10-03
Anticipated expiration: 2040-11-19
Also published as: CN113717256A

Abstract

The application relates to a fusion protein for preparing a single-cell in-situ active R-loop library and application thereof, wherein the fusion protein relates to an R-loop specific binding protein HBD, mnase nuclease and Tn5 transposase. The fusion protein is mainly used for R-loop detection and high-throughput library construction. The fusion protein is used for detecting R-loop and constructing a high-flux library, can realize in-situ active R-loop detection, can improve library construction efficiency, reduce library background, improve the accuracy of an R-loop detection technology, and simplify an R-loop detection flow.

Description

Fusion protein and application thereof

Technical Field

The application relates to the technical field of biology, in particular to a fusion protein and application thereof.

Background

R-loop (R-loop) is a three-stranded nucleic acid structure, i.e., one RNA strand is associated with one strand of double-stranded DNA and the other DNA strand is free to form a circular structure with the RNA-DNA hybrid strand. Previously, R-loop lengths were considered detrimental to cells and less interesting. In recent years, R-loop has been involved in many biological functions as an important element in the genome of cells, and has become an important field of research in epigenetics. Currently, most of the methods for detecting R-loop are based on DRIP-seq of antibody S9.6 and the derivation of DRIP-seq.

DRIP-seq ((DNA: RNA hybrid immunoprecipitation and sequencing) is an R-loop distribution method based on specific recognition of DNA: RNA hybrid strand antibodies and high throughput sequencing analysis technology, which has been used to detect the genome-wide level of human, mouse, yeast and the like, the flow path of DRIP-seq and DRIP-seq derivatization methods approximately comprises the steps of taking cells and other biological samples, extracting genomic DNA, cutting genomic DNA into DNA fragments of a certain size by restriction endonuclease, performing immunoprecipitation by antibody S9.6 and enriching the DNA fragments containing R-loop, and performing purification and recovery of DNA fragments, wherein the current DRIP-seq and DRIP-seq derivatization methods have the defects of limited resolution of detection signals and insufficient specificity of S9.6 antibodies, in particular S9.6 can recognize double-stranded RNA (dsRNA) and compare how to detect single cell-level rare R-loop activity.

In general, the existing R-loop detection method has some defects, mainly including: 1. the amount of the used cells is large; 2. not in situ; 3. lack of strand-specific information; 4. the process takes a long time.

However, the establishment of a novel single-cell activity R-loop detection method is of great importance for the study of the biological function of R-loop, and the key point is that a more suitable polypeptide is needed to enable the establishment of a novel single-cell activity R-loop detection method.

Disclosure of Invention

The application aims to provide a fusion protein for preparing a single-cell in-situ activity R-loop library, which can be used for a novel single-cell activity R-loop detection method and has the advantages of small required cell quantity, short detection time and the like.

The technical scheme for achieving the purpose is as follows.

A fusion protein comprises a dimer formed by a first functional region and a second functional region, wherein the first functional region comprises an R-loop specific binding protein HBD;

the second functional region comprises MNase nuclease or Tn5 transposase;

and (3) connecting the first functional area and the second functional area.

In some embodiments, the HBD is an R-loop specific recognition protein, the amino acid sequence of which is shown in SEQ ID No.1, or an amino acid sequence having the same function and having one or more amino acids substituted, deleted or added based on the sequence shown in SEQ ID No. 1.

In some embodiments, the MNase nuclease is a wild-type MNase truncate, and more preferably has an amino acid sequence shown in SEQ ID No.2, or is an amino acid sequence with the same function, wherein one or more amino acids are substituted, deleted or added on the basis of the sequence shown in SEQ ID No. 2.

In some embodiments, the Tn5 transposase is a wild type Tn5 transposase mutant, the amino acid sequence of which is shown in SEQ ID No.3, or an amino acid sequence with the same function that is substituted, deleted or added with one or more amino acids based on the sequence shown in SEQ ID No. 3.

In some of these embodiments, the amino acid sequence of the linker is shown in SEQ ID NO. 4.

In some embodiments, the kit further comprises a protein purification tag comprising a His tag, a GST tag, an MBP tag, a SUMO tag, or the like.

In some of these embodiments, the second functional region of the protein is linked to the N-terminus (nitrogen-terminus) of the amino acid functional region of the first functional region.

In some embodiments, the monomer of the fusion protein is HBD-MNase or HBD-Tn5, the amino acid sequence of the fusion protein HBD-MNase is shown as SEQ ID No.5, or one or more amino acids are substituted, deleted or added on the basis of the sequence shown as SEQ ID No.5, and the amino acid sequences have the same function; the amino acid sequence of the fusion protein HBD-Tn5 is shown as SEQ ID NO.6, or is an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids on the basis of the sequence shown as SEQ ID NO.6 and has the same function.

It is another object of the present application to provide the use of the fusion proteins described above for preparing R-loop high throughput sequencing libraries of biological samples.

In some of these embodiments, the fusion protein is used in the preparation of R-loop high throughput sequencing libraries of biological samples including, but not limited to, non-crosslinked, fixed or frozen/crosslinked, fixed or frozen cultured cell samples, tissue samples; the high-throughput sequencing library is a detection R-loop high-throughput sequencing library.

It is another object of the present application to provide a method for preparing a high throughput sequencing library of biological samples.

A method of preparing a high throughput sequencing library of biological samples comprising the steps of:

the method comprises the steps of collecting and processing biological samples to obtain single-cell suspension;

secondly, cleaning a biological sample by using a buffer solution, adding a proper amount of fusion protein, and cleaning unbound protein after the fusion protein is fully bound;

activating the fusion protein, and fully reacting to obtain small-fragment DNA or a DNA fragment with a label, wherein the small-fragment DNA is identified and cut by the fusion protein;

adding a stop solution to terminate the reaction, and purifying and recovering the DNA fragment;

and fifthly, performing PCR amplification to complete library construction.

The fusion protein is mainly used for constructing an R-loop high-throughput detection library, and compared with the prior method, the fusion protein has the following beneficial effects:

the application creatively connects proper MNase nuclease or Tn5 transposase with specific binding protein HBD through a linker (linker) to form a brand new fusion protein, and the fusion protein can obviously reduce the required cell quantity in the R-loop high-throughput detection process and even reach the single cell level: based on the traditional methods such as DRIP-seq, the cell demand needs to reach the level of tens of millions, but the detection method only needs hundreds of thousands of cells at most.

The fusion protein constructed by the application can obviously improve the specificity of detection on R-loop, the traditional detection mode DRIP-seq is to capture the R-loop in a fragmented genome by using an S9.6 antibody, but the specificity of the S9.6 antibody in the mode is not strong, the fusion protein can capture a hybrid chain in the R-loop and also can bind double-stranded RNA, so that the detected signal may not be real R-loop, RNaseH can specifically counteract the R-loop, the specific binding capacity of the RNaseH to the R-loop is hundreds of times stronger than that of the S9.6, and the specific binding capacity of the RNaseH to the R-loop is fused into fusion proteins HBD-Mnase and HBD-Tn5 of people by using RNaseH binding domain HBD, so that the specific recognition and capture of the R-loop are effectively improved.

Third, the fusion protein constructed by the application can remarkably simplify the experimental process, improve the library construction efficiency and reduce the library background in the R-loop high-throughput detection process: the library construction process of the application can be completed within five minutes at least by half an hour, and the library construction based on Tn5 can obviously reduce library background, tn5 in the fusion protein HBD-Tn5 can add specific adaptation at both ends of a cutting site while cutting genome, and after positive fragments after the adaptation is added are released, the library construction method of adding A and then the adaptation by conventional terminal repair is not required, library preparation can be directly and efficiently realized by PCR, the experimental process is obviously simplified, and the library construction efficiency is improved; namely, the fusion protein can rapidly realize the preparation of a single cell library with high flux in the high-flux detection of R-loop: the library construction flow of the detection method takes at most two hours, and the library construction based on HBD-Tn5 takes at least half an hour.

The fusion protein can be subjected to in-situ detection in the R-loop high-throughput detection process, so that the spatial in-situ information of a sample is effectively reserved. The detection of the application does not need the traditional chemical reagent treatment for crosslinking, does not need the traditional enzyme cutting or ultrasonic destroying subcellular structure, incubates the fusion protein related to the application with cells after cell perforation, and stores the fusion protein in Ca ²⁺ Or Mg (Mg) ²⁺ Under the action of the fusion protein, the fusion protein is activated to perform in-situ cleavage of R-loop and release of genome, and the most original structure of the active cells is maintained in the process. Whereas conventional DRIP-seq requires crosslinking of cells in tens of millions of cell amounts and requires genome fragmentation by digestion or ultrasound, resulting in loss of in situ information.

Drawings

Fig. 1: constructing a map of the HBD-Mnase expression vector.

Fig. 2: constructing a map of the HBD-Tn5 expression vector.

Fig. 3: high purity HBD-Mnase purification results.

Fig. 4: purification results of high purity HBD-Tn 5.

Fig. 5: HBD-Mnase activity detection results.

Fig. 6: HBD-Tn5 activity assay results.

Fig. 7: the HBD-Mnase fusion protein provided by the application is applied to R-mapping result analysis and track graph comparison with traditional DRIP-seq.

Fig. 8: the HBD-Tn5 fusion protein provided by the application is applied to R-mapping result analysis and track diagram comparison with a traditional detection method DRIP-seq.

Detailed Description

In order that the application may be understood more fully, a more particular description of the application will be rendered by reference to specific embodiments thereof which are illustrated in the appended claims. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It should be understood that the experimental methods in the following examples, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions suggested by the manufacturer. The various reagents commonly used in the examples are all commercially available products.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The application is further illustrated by the following specific examples, which are not intended to limit the scope of the application.

The fusion proteins constructed in accordance with the present application can be used for high throughput detection of R-loop in the following examples.

The fusion protein comprises a dimer formed by a first functional region and a second functional region, wherein the first functional region comprises an R-loop specific binding protein HBD, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1;

the second functional region comprises Mnase nuclease (the amino acid sequence of which is shown as SEQ ID NO. 2) and Tn5 transposase (the amino acid sequence of which is shown as SEQ ID NO. 3);

third, the linker structure (linker) connecting the first and second functional regions has an amino acid sequence shown in SEQ ID NO.4, and a protein purification Tag of 6 XHis Tag.

The monomer of the fusion protein is HBD-Mnase or HBD-Tn5, the amino acid sequence of the fusion protein HBD-Mnase is shown as SEQ ID NO.5, and the amino acid sequence of the fusion protein HBD-Tn5 is shown as SEQ ID NO. 6.

SEQ ID NO.1

>HBD

>MFYAVRRGRRTGVFLSWSECKAQVDRFPAARFKKFATEDEAWAF

SEQ ID NO.2

>MNase

>ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEAS AFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKP NNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ

SEQ ID NO.3

>Tn5

>MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEG AYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKS RGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGS MMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGY QISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWL LLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVA VRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAG SLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLDGFLAAKDLMAQGIKI

Linker1 (for HBD-Mnase)

>DDDKEF

Linker2 (for HBD-Tn 5)

>DDDKEFGGGGS(SEQ ID NO.4)

>6×His Tag

>HHHHHH

SEQ ID NO.5

>HBD-MNase

>MFYAVRRGRRTGVFLSWSECKAQVDRFPAARFKKFATEDEAWAFDDDKEFGGGGSATS TKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTK KMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTH EQHLRKSEAQAKKEKLNIWSEDNADSGQ

SEQ ID NO.6

>HBD-Tn5

>MFYAVRRGRRTGVFLSWSECKAQVDRFPAARFKKFATEDEAWAFDDDKEFGGGGSMIT SALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFI RNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGW WVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGSMMS NVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQISIP QKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTS EPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLL QLRESFTPPQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQW AYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLDGFLAAKDLMAQGIKI

Example 1

1. Design, expression and purification of HBD-Mnase fusion proteins

1. Construction of HBD-Mnase fusion protein expression vector

The method comprises the steps of respectively amplifying a first functional region (SEQ ID NO. 1) and a second functional region (SEQ ID NO. 2) by using primers. The primers were as follows:

first functional region forward primer:

ATGGGTCGCGGATCCGAATTCATGTTCTATGCGGTGAGGAG SEQ ID NO.7

first functional region reverse primer:

GAACTCCTTATCGTCATCAAAGGCCCAGGCCTCATCTT SEQ ID NO.8

second functional region forward primer:

GATGACGATAAGGAGTTCGCAACTTCAACTAAAAAATTACA SEQ ID NO.9

second functional region reverse primer:

GGTGGTGGTGGTGGTGCTCGAGTTATTGACCTGAATCAGCGTTGTC SEQ ID NO.10

secondly, amplifying the two DNA fragments into a fragment by using a bridge PCR (polymerase chain reaction) mode, namely amplifying a first functional region by using a forward primer and a reverse primer of the first functional region, and amplifying a second functional region by using a forward primer and a reverse primer of the second functional region; and then the PCR product of the first functional region and the PCR product of the second functional region are used as templates, and the forward primer of the first functional region and the reverse primer of the second functional region are used for amplifying the spliced fragments of the first functional region and the second functional region.

Cloning of the first functional region (template sequence Source: NCBI Reference Sequence: NM-011275.3):

PCR reaction System (50. Mu.L) (enzyme KOD-Plus Toyobo Cat#KOD-201 used):

10×KOD Plus Buffer 5μL，dNTP 4μL，Mg ₂ SO4 2μL，F+R Primer(10mM)2μL，template 1μL (500ng)，KOD Plus 1μL，ddH ₂ O 35μL；

the procedure is as follows: pre-denaturation at 95 ℃ for 3min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s; extending at 68 ℃ for 15s; extending at 68deg.C for 5min; preserving at 12 ℃; for a total of 35 cycles.

Cloning of the second functional region (template sequence Source: genBank: V01281.1):

PCR reaction system:

10×KOD Plus Buffer 5μL，dNTP 4μL，25mM Mg ₂ SO4 2μL，F+R Primer(10mM)2μL， template 1μL(500ng)，KOD-Plus 1μL，ddH ₂ O 35μL；

the procedure is as follows: pre-denaturation at 95 ℃ for 3min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s; extending at 68 ℃ for 30s; extending at 68deg.C for 5min; preserving at 12 ℃; for a total of 35 cycles.

PCR products were detected by 1% agarose gel electrophoresis, and the target fragment was recovered using Biospin Gel Extraction Kit gel recovery kit (BIO FLUX, cat#BSC02M1)).

Bridge PCR reaction system:

10×KOD Plus Buffer 5μL，dNTP 4μL，25mM Mg ₂ SO4 2. Mu.L, F+R Primer (10 mM) 2. Mu.L, template 2. Mu.L (molar ratio of two functional fragments 1:1), KOD-Plus 1. Mu.L, ddH ₂ O 34μL；

The procedure is as follows: pre-denaturation at 95 ℃ for 3min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s; extending at 68 ℃ for 40s; extending at 68deg.C for 5min; stored at 12℃for a total of 35 cycles.

The PCR products were detected by 1% agarose gel electrophoresis, biospin Gel Extraction Kit gel recovery kit (BIO FLUX,

cat#bsc02m1)) to recover the fragment of interest.

(3) The fragment was cloned into expression vector pET-28a as shown in FIG. 1.

Adopts a mode of homologous recombination, and a reaction system:

pET-28a ((EcoRI+Xhol double cleavage product) 20ng, 60ng of the desired fragment recovered from the reaction mixture, 5 XLication-Free Cloning 2. Mu.L (ABM), ddH ₂ O was made up to 10. Mu.L and ice-bathed for 30min. The homologous recombination products were transformed into DH 5. Alpha (Trans).

Sanger sequencing ensures the integrity of the vector sequence.

The monoclonal was sequenced unidirectionally with T7 promoter.

2. Expression and purification of HBD-Mnase fusion proteins

The HBD-Mnase fusion protein expression plasmid with correct sequence is transferred into BL21 (DE 3) expression strain.

A monoclonal strain expressing the HBD-Mnase fusion protein is inoculated in LB culture medium containing kanamycin, and cultured at 37 ℃ and 220rpm overnight.

Third step the cultures in the second step were inoculated into 100mL of LB medium, 37 ℃,220rpm, and cultivated for 3h to od=0.6-0.8. The culture flask of the third step was placed in a refrigerator at 4℃for 15min, and IPTG was added to a final concentration of 0.5mM.

And fifthly, culturing at 20 ℃ and 160rpm for 2 hours, collecting thalli, and performing ultrasonic or high-pressure crushing to obtain a protein solution.

Sixthly, centrifuging the protein solution obtained in step five at 4 ℃ for 30min with 13000g, precipitating bacterial genome with PEI (final concentration of 0.05%), centrifuging for 30min with 13000g at 4 ℃, and filtering the supernatant with a 0.45 μm filter head.

The Ni column was first equilibrated using Ni column affinity purification (Ni-NTA 1ml Pre-Packed Gravity Column, protect#C600791-0010) and after Buffer flow in the Ni column was completed, pre-chilled 20mM imidozole (formulated in 50mM Tris-HCl, pH= 7.5,0.8M NaCl,0.2%Triton X-100 and 10% glychol) was added to equilibrate the Ni column. Thereafter, the protein solution was repeatedly applied to the column 5 times, unbound protein was washed off with 150mL of pre-chilled 20mM imidozole in multiple washes, the column was washed with 10mL 50mM imidazole, and finally the protein of interest was eluted with 5mL of pre-chilled 300mM imidozole. The eluted target protein was dialyzed in a dialysate (20 mM Tris-HCl, pH=7.5, 150mM NaCl,10% glycerol) at 4℃overnight. The HBD-Mnase was concentrated using a 10kDa protein concentration column and the BCA method was used to detect protein concentration after concentration while the Qubit detected residual bacterial genome (the residual genome amount was controlled to be less than 0.5 ng/. Mu.L).

The purified proteins were subjected to SDS-PAGE and Coomassie blue staining as shown in FIG. 3. FT represents the flow-through of the protein on the column, imidozole represents the elution products of imidazole eluents with different concentrations (50 mM imidozole represents washing off nonspecific or poorly bound proteins, 300mM imidozole represents the final eluted target protein solution), M represents the protein marker, and meanwhile, the red asterisk marked band in the 300mM imidozole lane is the purified target protein product. The amino acid sequence of the HBD-Mnase fusion protein is shown as SEQ ID NO. 5. The function of the HBD-Tn5 fusion protein can be realized by a person skilled in the art by substituting, deleting or adding one or more amino acids based on the sequence shown in SEQ ID NO.5 according to common knowledge of the person skilled in the art and the amino acid sequence with the same function.

Example 2 design, expression and purification of HBD-Tn5 fusion proteins

1. Construction of HBD-Tn5 fusion protein expression vector

The method comprises the steps of respectively amplifying a first functional region (SEQ ID NO. 1) and a second functional region (SEQ ID NO. 3) by using primers. The primers were as follows:

first functional region forward primer:

ATGGGTCGCGGATCCGAATTCATGTTCTATGCGGTGAGGAG SEQ ID NO.7

first functional region reverse primer:

GGCTTTAGCCGCTGCCTCCTTTGCGGCAGCAAAGGCCCAGGCCTCATCTT SEQ ID NO.11

second functional region forward primer:

GCTGCCGCAAAGGAGGCAGCGGCTAAAGCCATGATTACCAGTGCACTGCA SEQ ID NO.12

second functional region reverse primer:

GGTGGTGGTGGTGGTGCTCGAGTTAGATTTTAATGCCCTGCGCCATC SEQ ID NO.13

Cloning the first functional region:

PCR reaction System (50. Mu.L) template sequence Source: NCBI Reference Sequence NM-011275.3: 10 XKOD Plus Buffer 5. Mu.L, dNTP 4. Mu.L, 25mM Mg ₂ SO4 2μL，F+R Primer(10mM)2μL， template 1μL(500ng)，KOD Plus 1μL，ddH ₂ O 35μL；

The procedure is as follows: pre-denaturation at 95 ℃ for 3min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s; extending at 68 ℃ for 15s; extending at 68deg.C for 5min; stored at 12℃for a total of 35 cycles.

Cloning the second functional region:

PCR reaction system:

the procedure is as follows: pre-denaturation at 95 ℃ for 3min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s; extending at 68 ℃ for 1min for 30s; extending at 68deg.C for 5min; stored at 12℃for a total of 35 cycles.

Bridge PCR reaction System (enzyme KOD-Plus Toyobo Cat#KOD-201 used):

The procedure is as follows: pre-denaturation at 95 ℃ for 3min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s; extending at 68℃for 1min 45s; extending at 68deg.C for 5min; stored at 12℃for a total of 35 cycles.

Third, the fragment was cloned into the expression vector pET-28a, as shown in FIG. 2.

Adopts a mode of homologous recombination, and a reaction system:

pET-28a (EcoRI+Xhol double cleavage product) 20ng; 60ng of the target fragment obtained by recycling in the second step; 5 Xligation-Free Cloning 2. Mu.L (ABM); ddH ₂ O was made up to 10. Mu.L and ice-bathed for 30min. The homologous recombination product was transformed into DH 5. Alpha (TransGen).

Sanger sequencing ensures the integrity of the vector sequence.

The monoclonal was sequenced bi-directionally with T7promoter and T7 terminator.

2. Expression and purification of HBD-Tn5 fusion proteins

The HBD-Tn5 fusion protein expression plasmid with correct sequence is transferred into BL21 (DE 3) expression strain.

A monoclonal strain expressing the HBD-Tn5 fusion protein is inoculated in LB culture medium containing kanamycin, and cultured at 37 ℃ and 220rpm overnight.

Third step the cultures in the second step were inoculated into 100mL of LB medium, 37 ℃,220rpm, and cultivated for 3h to od=0.6-0.8.

The culture flask of the third step was placed in a refrigerator at 4℃for 15min, and IPTG was added to a final concentration of 0.5mM.

And fifthly, culturing at 37 ℃ and 160rpm for 2 hours, collecting thalli, and carrying out ultrasonic or high-pressure crushing to obtain a protein solution.

Sixthly, centrifuging 13000g of the protein solution obtained in step five at 4 ℃ for 30min, precipitating bacterial genome by using PEI (final concentration is 0.05%) of the protein solution obtained in step five, centrifuging 13000g of the protein solution at 4 ℃ for 30min, and filtering by using a 0.45 mu m filter head.

Affinity purification of Ni column

The nickel column was equilibrated first, and after Buffer run out in the Ni column, pre-chilled 20mM imidozole (formulated in 50mM Tris-HCl, pH= 7.5,0.8M NaCl,0.2%Triton X-100 and 10% glychol) was added to equilibrate the nickel column. Thereafter, the protein was repeatedly applied to the column 5 times, washed with 150mL of a pre-chilled 20mM imidozole fraction multiple times, then 10mL 35mM imidazole, and finally eluted with 5mL of a pre-chilled 300mM imidozole. The eluted protein was dialyzed directly in buffer (20 mM Tris-HCl, pH=7.5, 150mM NaCl,10%glycerol) at 4℃overnight. The protein concentration was measured by the BCA method after concentration by concentrating HBD-Tn5 using a 30kDa protein concentration column, while the residual bacterial genome was measured by Qubit (the residual genome amount was controlled to be less than 0.5 ng/. Mu.L).

The purified proteins were subjected to SDS-PAGE and Coomassie blue staining as shown in FIG. 4. Lanes FT represent the flow-through, imidazole represents the eluted product of imidazole eluents at different concentrations (35 mM imidazole represents washing away unbound protein, 300mM imidazole represents final eluted protein solution of interest), M represents protein marker, and the red asterisk marked band in 300mM imidazole lanes is the purified protein product of interest.

The amino acid sequence of the HBD-Tn5 fusion protein is shown as SEQ ID NO. 6. The function of the HBD-Tn5 fusion protein can be realized by a person skilled in the art by substituting, deleting or adding one or more amino acids based on the sequence shown in SEQ ID NO.6 according to common knowledge of the person skilled in the art and the amino acid sequence with the same function.

Example 3 detection of HBD-Mnase Activity

Different amounts of HBD-Mnase (the amount added is shown in FIG. 5) were mixed with 550ng of genomic DNA and Ca was added ²⁺ The final concentration was 10mM and the reaction was carried out in an ice bath for 10min. Agarose gel electrophoresis detection shows that HBD-Mnase can cleave genomic DNA into DNA fragments of 100bp in size, and the results are shown in FIG. 5: with the same genome input (550 ng), the gradually narrowing and even disappearance of the genome band can be obviously seen along with the increase of the fusion protein HBD-Mnase input (from 0ng to 578.4 ng), and the gradually increasing and diffuse genome appears below the lanes, so that the in vitro enzyme digestion gene results show that the fusion protein HBD-Mnase has higher enzyme activity.

Example 4 detection of HBD-Tn5 Activity

15pmol of HBD-Tn5 was mixed with 200ng of genomic DNA and Mg was added ²⁺ The final concentration was 10mM, reacted at 55℃for 10min, and subjected to agarose gel electrophoresis detection as shown in FIG. 6: compared with a control group without HBD-Tn5 treatment, the addition of HBD-Tn5 can effectively break genomic DNA, so that obvious genomic strips disappear, diffuse genomic distribution appears below a lane, most of fragment distribution is below 1000bp, and in-vitro enzyme digestion genome experiments prove that the fusion protein HBD-Tn5 has higher enzyme activity.

Example 5

The HBD-Mnase and HBD-Tn5 obtained by the application are respectively applied to non-crosslinked cells in a natural state, and R-loop is detected in situ, and the detection method comprises the following steps:

collecting 100000 in vitro cultured HEK 293T cells, washing with PBS (PH=7.2), centrifuging at 600g at room temperature for 3min, removing supernatant, washing with 200 mu L of wash buffer (10 mM HEPES (4-hydroxyethyl piperazine ethane sulfonic acid), 150mM NaCl, 0.5mM spermidine; 5% digitonin);

a binding buffer (10mM HEPES,10mM KCl,1mM CaCl) is carried out on 8 mu L of Con A beads (Bangslabs) ₂ ，1mM MnCl ₂ ) After washing, mixing and incubating with the first medium cells for 15min, and centrifuging at 4 ℃ to obtain 100gInstantaneously separating, discarding the supernatant (a magnetic frame), adding fusion protein HBD-Mnase or HBD-Tn5 into 100 mu L of wash buffer, wherein the final concentration is 1 mu M,5% Digitonin (Digitonin), 1 mu L of PIC (100X), and incubating for 2-12 h at 4 ℃;

thirdly, washing with 200 mu L of wash buffer for three times, washing off superfluous protein, performing instantaneous separation on 100g of the centrifuge at 4 ℃, and discarding the supernatant (a magnetic rack);

adding 10mM Ca into the four sides ²⁺ Or Mg (Mg) ²⁺ Activating fusion protein HBD-MNase or HBD-Tn5 to cut DNA (R-mapping), wherein the reaction condition of the fusion protein HBD-MNase is 0+/-0.5 ℃ for 30min, and the reaction condition of the fusion protein HBD-Tn5 is 37-55 ℃ for 1 h);

after the cleavage reaction was terminated by addition of 10mM EDTA, phenol: chloroform: extracting DNA from isoamyl alcohol;

sixth, based on HBD-MNase (R-mapping) library construction, the extracted genome fragment is subjected to end repair and adapter (Vazyme VAHTS Adapter-S for illumine) is connected, and PCR library construction is performed (Vazyme index for illumina).

PCR system: 24. Mu.L of DNA, 1. Mu. L i5 (10 mM), 1. Mu. L i7 (10 mM), 10. Mu.L of 5X KAPA HiFi Fidelity buffer, 1. Mu. L KAPA HiFi hot start, 11.5. Mu.L of ddH ₂ O, 1.5. Mu.L dNTP; and a total of 50. Mu.L.

The procedure is as follows: pre-denaturation at 98 ℃ for 45s; denaturation at 98℃for 15s; annealing at 60 ℃ for 30s; extending at 72deg.C for 1min;72 ℃, total extension 1min,12 ℃, preservation, 13 cycles total; the library products were sequenced by illuminea.

And (5) PCR-pooling of the purified genome based on HBD-Tn5 (R-mapping) (Vazyme index for illumina).

PCR system: 23. Mu.L of DNA, 1. Mu. L i5 (10 mM), 1. Mu. L i7 (10 mM), 25. Mu.L of 2 XMix buffer (NEB Cat#M0541S); the procedure is as follows: 72 ℃ for 5min; pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 15s; annealing at 60 ℃ for 30s; extending at 72deg.C for 1min;72 ℃ and extending for 5min at 16 ℃ and preserving for 13 cycles; the library products were submitted to illuminea sequencing.

Biological samples for detection of fusion proteins of the present application may include, but are not limited to, cultured cell samples, tissue samples or other biological samples that are not crosslinked, fixed or frozen/crosslinked, fixed or frozen.

PCR pooling can be accomplished by using commercially available kits or methods to obtain DNA, and using other polymerases, including isothermal polymerases and other polymerases with strand displacement properties, RNA or DNA dependent polymerases for PCR amplification and pooling.

As shown in fig. 7 and 8: the results obtained by applying the fusion proteins HBD-Mnase and HBD-Tn5 of the present application to in situ detection of R-loop (R-mapping) were compared with the results of the conventional method for detecting R-loop DRIP-seq (R-loop formation is a distinctive characteristic of unmethylated human CpG island pro ters. Ginno et al, mol cell. 2012Mar 30;45 (6): 814-25).

As shown in fig. 7: under the condition of relatively less cell quantity, the R-mapping based on HBD-MNase can capture the same positive signal of DRIP-seq, the peak signal of the application on the gene locus is more concentrated, the DRIP-seq signal is relatively more dispersed, and the signal value of the R-mapping detection method is also higher, which shows that the signal to noise ratio (on figure 7) of the application (based on the fusion protein HBD-MNase) is effectively improved compared with the traditional method. Meanwhile, the track diagram also shows that the application can detect the signal which cannot be captured by the DRIP-seq (in fig. 7), and the signal which is specifically detected by the application can be resolved after RNase H treatment (in fig. 7), which shows that the signal is real R-loop, and the application is proved to be capable of effectively realizing the accurate capture of the R-loop compared with the traditional method;

the application is based on the application of the HBD-Tn5 fusion protein, not only has less cell consumption, but also can greatly shorten the library construction time to within half an hour based on the characteristic that Tn5 is directly connected with an adapter after genome is cut and then PCR library construction is carried out.

As shown in fig. 8: the application is based on the comparison and analysis of the results of in-situ detection of R-loop (on figure 8) and the DRIP-seq (under figure 8) of traditional detection of R-loop in non-crosslinked cells of the application of the R-mapping of HBD-Tn5 fusion protein, the R-loop peak signal obtained by the method for detecting the fusion protein is more concentrated and relatively more accurate, the DRIP-seq signal is relatively more dispersed, and the signal value obtained by the method is higher, namely, compared with the traditional DRIP-seq, the application shows that the detection of the in-situ R-loop by the HBD-Tn5 can effectively improve the signal to noise ratio.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

Sequence listing

<110> Guangzhou biomedical and health institute of China academy of sciences

<120> a fusion protein and use thereof

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<170> SIPOSequenceListing 1.0

<210> 1

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<212> PRT

<213> Artificial sequence (Artificial Sequence)

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Met Phe Tyr Ala Val Arg Arg Gly Arg Arg Thr Gly Val Phe Leu Ser

1 5 10 15

Trp Ser Glu Cys Lys Ala Gln Val Asp Arg Phe Pro Ala Ala Arg Phe

20 25 30

Lys Lys Phe Ala Thr Glu Asp Glu Ala Trp Ala Phe

35 40

<210> 2

<211> 149

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Ala Thr Ser Thr Lys Lys Leu His Lys Glu Pro Ala Thr Leu Ile Lys

1 5 10 15

Ala Ile Asp Gly Asp Thr Val Lys Leu Met Tyr Lys Gly Gln Pro Met

20 25 30

Thr Phe Arg Leu Leu Leu Val Asp Thr Pro Glu Thr Lys His Pro Lys

35 40 45

Lys Gly Val Glu Lys Tyr Gly Pro Glu Ala Ser Ala Phe Thr Lys Lys

50 55 60

Met Val Glu Asn Ala Lys Lys Ile Glu Val Glu Phe Asp Lys Gly Gln

65 70 75 80

Arg Thr Asp Lys Tyr Gly Arg Gly Leu Ala Tyr Ile Tyr Ala Asp Gly

85 90 95

Lys Met Val Asn Glu Ala Leu Val Arg Gln Gly Leu Ala Lys Val Ala

100 105 110

Tyr Val Tyr Lys Pro Asn Asn Thr His Glu Gln His Leu Arg Lys Ser

115 120 125

Glu Ala Gln Ala Lys Lys Glu Lys Leu Asn Ile Trp Ser Glu Asp Asn

130 135 140

Ala Asp Ser Gly Gln

145

<210> 3

<211> 476

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Met Ile Thr Ser Ala Leu His Arg Ala Ala Asp Trp Ala Lys Ser Val

1 5 10 15

Phe Ser Ser Ala Ala Leu Gly Asp Pro Arg Arg Thr Ala Arg Leu Val

20 25 30

Asn Val Ala Ala Gln Leu Ala Lys Tyr Ser Gly Lys Ser Ile Thr Ile

35 40 45

Ser Ser Glu Gly Ser Lys Ala Met Gln Glu Gly Ala Tyr Arg Phe Ile

50 55 60

Arg Asn Pro Asn Val Ser Ala Glu Ala Ile Arg Lys Ala Gly Ala Met

65 70 75 80

Gln Thr Val Lys Leu Ala Gln Glu Phe Pro Glu Leu Leu Ala Ile Glu

85 90 95

Asp Thr Thr Ser Leu Ser Tyr Arg His Gln Val Ala Glu Glu Leu Gly

100 105 110

Lys Leu Gly Ser Ile Gln Asp Lys Ser Arg Gly Trp Trp Val His Ser

115 120 125

Val Leu Leu Leu Glu Ala Thr Thr Phe Arg Thr Val Gly Leu Leu His

130 135 140

Gln Glu Trp Trp Met Arg Pro Asp Asp Pro Ala Asp Ala Asp Glu Lys

145 150 155 160

Glu Ser Gly Lys Trp Leu Ala Ala Ala Ala Thr Ser Arg Leu Arg Met

165 170 175

Gly Ser Met Met Ser Asn Val Ile Ala Val Cys Asp Arg Glu Ala Asp

180 185 190

Ile His Ala Tyr Leu Gln Asp Lys Leu Ala His Asn Glu Arg Phe Val

195 200 205

Val Arg Ser Lys His Pro Arg Lys Asp Val Glu Ser Gly Leu Tyr Leu

210 215 220

Tyr Asp His Leu Lys Asn Gln Pro Glu Leu Gly Gly Tyr Gln Ile Ser

225 230 235 240

Ile Pro Gln Lys Gly Val Val Asp Lys Arg Gly Lys Arg Lys Asn Arg

245 250 255

Pro Ala Arg Lys Ala Ser Leu Ser Leu Arg Ser Gly Arg Ile Thr Leu

260 265 270

Lys Gln Gly Asn Ile Thr Leu Asn Ala Val Leu Ala Glu Glu Ile Asn

275 280 285

Pro Pro Lys Gly Glu Thr Pro Leu Lys Trp Leu Leu Leu Thr Ser Glu

290 295 300

Pro Val Glu Ser Leu Ala Gln Ala Leu Arg Val Ile Asp Ile Tyr Thr

305 310 315 320

His Arg Trp Arg Ile Glu Glu Phe His Lys Ala Trp Lys Thr Gly Ala

325 330 335

Gly Ala Glu Arg Gln Arg Met Glu Glu Pro Asp Asn Leu Glu Arg Met

340 345 350

Val Ser Ile Leu Ser Phe Val Ala Val Arg Leu Leu Gln Leu Arg Glu

355 360 365

Ser Phe Thr Pro Pro Gln Ala Leu Arg Ala Gln Gly Leu Leu Lys Glu

370 375 380

Ala Glu His Val Glu Ser Gln Ser Ala Glu Thr Val Leu Thr Pro Asp

385 390 395 400

Glu Cys Gln Leu Leu Gly Tyr Leu Asp Lys Gly Lys Arg Lys Arg Lys

405 410 415

Glu Lys Ala Gly Ser Leu Gln Trp Ala Tyr Met Ala Ile Ala Arg Leu

420 425 430

Gly Gly Phe Met Asp Ser Lys Arg Thr Gly Ile Ala Ser Trp Gly Ala

435 440 445

Leu Trp Glu Gly Trp Glu Ala Leu Gln Ser Lys Leu Asp Gly Phe Leu

450 455 460

Ala Ala Lys Asp Leu Met Ala Gln Gly Ile Lys Ile

465 470 475

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Asp Asp Asp Lys Glu Phe Gly Gly Gly Gly Ser

1 5 10

<210> 5

<211> 204

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Met Phe Tyr Ala Val Arg Arg Gly Arg Arg Thr Gly Val Phe Leu Ser

1 5 10 15

Trp Ser Glu Cys Lys Ala Gln Val Asp Arg Phe Pro Ala Ala Arg Phe

20 25 30

Lys Lys Phe Ala Thr Glu Asp Glu Ala Trp Ala Phe Asp Asp Asp Lys

35 40 45

Glu Phe Gly Gly Gly Gly Ser Ala Thr Ser Thr Lys Lys Leu His Lys

50 55 60

Glu Pro Ala Thr Leu Ile Lys Ala Ile Asp Gly Asp Thr Val Lys Leu

65 70 75 80

Met Tyr Lys Gly Gln Pro Met Thr Phe Arg Leu Leu Leu Val Asp Thr

85 90 95

Pro Glu Thr Lys His Pro Lys Lys Gly Val Glu Lys Tyr Gly Pro Glu

100 105 110

Ala Ser Ala Phe Thr Lys Lys Met Val Glu Asn Ala Lys Lys Ile Glu

115 120 125

Val Glu Phe Asp Lys Gly Gln Arg Thr Asp Lys Tyr Gly Arg Gly Leu

130 135 140

Ala Tyr Ile Tyr Ala Asp Gly Lys Met Val Asn Glu Ala Leu Val Arg

145 150 155 160

Gln Gly Leu Ala Lys Val Ala Tyr Val Tyr Lys Pro Asn Asn Thr His

165 170 175

Glu Gln His Leu Arg Lys Ser Glu Ala Gln Ala Lys Lys Glu Lys Leu

180 185 190

Asn Ile Trp Ser Glu Asp Asn Ala Asp Ser Gly Gln

195 200

<210> 6

<211> 531

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Met Phe Tyr Ala Val Arg Arg Gly Arg Arg Thr Gly Val Phe Leu Ser

1 5 10 15

Trp Ser Glu Cys Lys Ala Gln Val Asp Arg Phe Pro Ala Ala Arg Phe

20 25 30

Lys Lys Phe Ala Thr Glu Asp Glu Ala Trp Ala Phe Asp Asp Asp Lys

35 40 45

Glu Phe Gly Gly Gly Gly Ser Met Ile Thr Ser Ala Leu His Arg Ala

50 55 60

Ala Asp Trp Ala Lys Ser Val Phe Ser Ser Ala Ala Leu Gly Asp Pro

65 70 75 80

Arg Arg Thr Ala Arg Leu Val Asn Val Ala Ala Gln Leu Ala Lys Tyr

85 90 95

Ser Gly Lys Ser Ile Thr Ile Ser Ser Glu Gly Ser Lys Ala Met Gln

100 105 110

Glu Gly Ala Tyr Arg Phe Ile Arg Asn Pro Asn Val Ser Ala Glu Ala

115 120 125

Ile Arg Lys Ala Gly Ala Met Gln Thr Val Lys Leu Ala Gln Glu Phe

130 135 140

Pro Glu Leu Leu Ala Ile Glu Asp Thr Thr Ser Leu Ser Tyr Arg His

145 150 155 160

Gln Val Ala Glu Glu Leu Gly Lys Leu Gly Ser Ile Gln Asp Lys Ser

165 170 175

Arg Gly Trp Trp Val His Ser Val Leu Leu Leu Glu Ala Thr Thr Phe

180 185 190

Arg Thr Val Gly Leu Leu His Gln Glu Trp Trp Met Arg Pro Asp Asp

195 200 205

Pro Ala Asp Ala Asp Glu Lys Glu Ser Gly Lys Trp Leu Ala Ala Ala

210 215 220

Ala Thr Ser Arg Leu Arg Met Gly Ser Met Met Ser Asn Val Ile Ala

225 230 235 240

Val Cys Asp Arg Glu Ala Asp Ile His Ala Tyr Leu Gln Asp Lys Leu

245 250 255

Ala His Asn Glu Arg Phe Val Val Arg Ser Lys His Pro Arg Lys Asp

260 265 270

Val Glu Ser Gly Leu Tyr Leu Tyr Asp His Leu Lys Asn Gln Pro Glu

275 280 285

Leu Gly Gly Tyr Gln Ile Ser Ile Pro Gln Lys Gly Val Val Asp Lys

290 295 300

Arg Gly Lys Arg Lys Asn Arg Pro Ala Arg Lys Ala Ser Leu Ser Leu

305 310 315 320

Arg Ser Gly Arg Ile Thr Leu Lys Gln Gly Asn Ile Thr Leu Asn Ala

325 330 335

Val Leu Ala Glu Glu Ile Asn Pro Pro Lys Gly Glu Thr Pro Leu Lys

340 345 350

Trp Leu Leu Leu Thr Ser Glu Pro Val Glu Ser Leu Ala Gln Ala Leu

355 360 365

Arg Val Ile Asp Ile Tyr Thr His Arg Trp Arg Ile Glu Glu Phe His

370 375 380

Lys Ala Trp Lys Thr Gly Ala Gly Ala Glu Arg Gln Arg Met Glu Glu

385 390 395 400

Pro Asp Asn Leu Glu Arg Met Val Ser Ile Leu Ser Phe Val Ala Val

405 410 415

Arg Leu Leu Gln Leu Arg Glu Ser Phe Thr Pro Pro Gln Ala Leu Arg

420 425 430

Ala Gln Gly Leu Leu Lys Glu Ala Glu His Val Glu Ser Gln Ser Ala

435 440 445

Glu Thr Val Leu Thr Pro Asp Glu Cys Gln Leu Leu Gly Tyr Leu Asp

450 455 460

Lys Gly Lys Arg Lys Arg Lys Glu Lys Ala Gly Ser Leu Gln Trp Ala

465 470 475 480

Tyr Met Ala Ile Ala Arg Leu Gly Gly Phe Met Asp Ser Lys Arg Thr

485 490 495

Gly Ile Ala Ser Trp Gly Ala Leu Trp Glu Gly Trp Glu Ala Leu Gln

500 505 510

Ser Lys Leu Asp Gly Phe Leu Ala Ala Lys Asp Leu Met Ala Gln Gly

515 520 525

Ile Lys Ile

530

<210> 7

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

atgggtcgcg gatccgaatt catgttctat gcggtgagga g 41

<210> 8

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gaactcctta tcgtcatcaa aggcccaggc ctcatctt 38

<210> 9

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gatgacgata aggagttcgc aacttcaact aaaaaattac a 41

<210> 10

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ggtggtggtg gtggtgctcg agttattgac ctgaatcagc gttgtc 46

<210> 11

<211> 50

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ggctttagcc gctgcctcct ttgcggcagc aaaggcccag gcctcatctt 50

<210> 12

<211> 50

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gctgccgcaa aggaggcagc ggctaaagcc atgattacca gtgcactgca 50

<210> 13

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

ggtggtggtg gtggtgctcg agttagattt taatgccctg cgccatc 47

Claims

1. A fusion protein is characterized in that a monomer of the fusion protein is HBD-Mnase, and the amino acid sequence of the fusion protein HBD-Mnase is shown as SEQ ID NO. 5.

2. A fusion protein is characterized in that a monomer of the fusion protein is HBD-Tn5, and the amino acid sequence of the fusion protein HBD-Tn5 is shown as SEQ ID NO. 6.

3. Use of the fusion protein of claim 1 for preparing an R-loop high throughput sequencing library of a biological sample.

4. Use of the fusion protein of claim 2 for preparing an R-loop high throughput sequencing library of a biological sample.

5. Use of the fusion protein of claim 1 for in situ active R-loop detection for non-disease diagnosis purposes.

6. Use of the fusion protein of claim 2 for in situ active R-loop detection for non-disease diagnosis purposes.