WO2020133590A1 - Method and test kit for quickly preparing monomolecular optical spectrum labeling library - Google Patents

Abstract

Provided is a method for quickly preparing a monomolecular optical spectrum labeling library, comprising the following steps: (1) extracting long genomic DNA; (2) labelling the extracted genomic DNA; (3) purifying the labeled genomic DNA; and (4) staining the backbone of the genomic DNA to obtain a monomolecular optical spectrum labeling library. Also provided is a test kit for preparing the monomolecular optical spectrum labeling library.

Description

Method and kit for quickly preparing single-molecule optical map labeling library Technical field

The invention relates to a method for quickly preparing a single molecule optical spectrum labeling library, and a kit suitable for the method.

Background technique

Many human diseases are caused by structural changes in chromosomes. At present, the commonly used methods of chromosome structure analysis include karyotype analysis and chip hybridization. However, these technologies have certain limitations, such as low resolution, high cost, and inability to cover the entire genome. The development of long-segment single-molecule optical spectroscopy technology makes up for the limitations of the above technology. BioNano introduced and the Irys Saphyr system using restriction enzymes or nicking of DNA methyltransferase, and to identify ^a fluorescent marker, again using the single DNA molecule nanochannel linear deployment, a high resolution and long monomolecular Fluorescence imaging can generate a map of specific enzyme site distribution. The optical single molecule technology can assist map genome assembly, disease detection, and detecting genetic structural chromosome ^2. However, the preparation of single-molecule optical map marker libraries at this stage has the disadvantages of time-consuming and cumbersome preparation process, and is not suitable for clinical detection.

In addition, the advantage of optical mapping technology is that it can label DNA at the single molecule level, so the length of genomic DNA fragments is one of the important factors that determine the effect of optical mapping. Generally, the average length of DNA fragments is greater than 200Kb, and the longest DNA fragment reaches Mb level. At present, the solid-phase method based on low-melting agarose gel is generally used to obtain long-length genomic DNA ^3-6 used for preparing optical map marker libraries. This method can extract high-purity genomic DNA up to Mb level, but includes steps such as embedding cells into agarose gel, proteinase K digestion, gel melting, gel digestion and genomic DNA dialysis. The operation process is very cumbersome , Which took more than 24 hours. In addition, the yield of long-length genomic DNA extracted by this method is often unstable, very viscous, and poor uniformity makes it difficult to measure the concentration, which affects the quality of subsequent optical map markers. This solid phase long fragment genomic DNA extraction coupled with BioNano's Direct Labeling and Staining (DLS) makes the preparation of optical map labeling libraries a total of 3.5 days. These factors are not conducive to the large-scale promotion and use of optical atlas technology.

In order to solve the above problems, the present invention provides a liquid phase system-based method for quickly preparing an optical map labeling library, which only includes four steps of extracting long genomic DNA, labeling genomic DNA, purifying labeled genomic DNA and staining genomic DNA . This method simplifies the process of optical atlas labeling, shortening the time from the original 3.5 days to 8 hours, and the resulting library is of high quality and strong reproducibility, which is beneficial to the application of single-molecule optical atlas technology in clinical detection.

Summary of the invention

The purpose of the present invention is to solve the problems of long time-consuming and cumbersome process for preparation of single-molecule optical spectrum library at the present stage. By extracting long-length genomic DNA in liquid phase and simplifying the single-molecule optical map labeling process, the present invention can realize the rapid preparation of a rapid single-molecule optical map labeling library.

Therefore, in the first aspect, the present invention provides a method for quickly preparing a single-molecule optical map labeling library, including the following steps:

(1) Extract long genomic DNA;

(2) Mark the extracted genomic DNA;

(3) Purification of labeled genomic DNA, and

(4) Stain the skeleton of genomic DNA to obtain a library of single-molecule optical map markers.

In one embodiment, the steps of extracting long fragments of genomic DNA include cell lysis, genomic DNA precipitation, genomic DNA washing, and genomic DNA lysis. In a preferred embodiment, the step of extracting long genomic DNA includes the following sub-steps:

a) Add lysate and proteinase K to the sample to lyse the cells and release genomic DNA;

b) Add a precipitant to the reaction system of step a) to obtain a precipitate of genomic DNA;

c) Wash the resulting genomic DNA precipitate with a cleaning solution;

d) Dissolve the genomic DNA in the dissolving solution.

In a preferred embodiment, the step of extracting long fragments of genomic DNA consists of the above four sub-steps a)-d).

In this context, "long-length genomic DNA" refers to genomic DNA with an average length greater than 200Kb and up to Mb level.

In one embodiment, the sample may be any tissue or cell of animal origin, such as cells from embryonic tissue, liver tissue, thymus tissue, spleen tissue, body fluids, or cell lines cultured in vitro, etc. Examples of body fluids include, but are not limited to, blood, serum, plasma, joint fluid, semen, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural fluid, bile, pancreatic fluid, and the like.

In one embodiment, the lysate contains NaCl, Tris-HCl (pH 7.4-8.0), EDTA (pH 8.0) and SDS. In another embodiment, the lysate consists of NaCl, Tris-HCl (pH 7.4-8.0), EDTA (pH 8.0) and SDS. The concentration of NaCl, Tris-HCl (pH7.4-8.0) and EDTA (pH8.0) in the lysate can be adjusted and determined by routine experiments according to specific experimental requirements. For example, the concentration of NaCl is 0.1M-0.5M, Tris -The concentration of HCl (pH7.4-8.0) is 10mM-200mM, and the concentration of EDTA (pH8.0) is 10mM-100mM. However, the concentration of SDS is critical to the quality of extracted genomic DNA. Specifically, if the concentration of SDS is too low, for example, less than 0.1%, it will result in insufficient cell lysis; if the concentration of SDS is too high, for example, higher than 8%, excessive SDS will precipitate with genomic DNA, affecting subsequent experiments . Therefore, in the present invention, the concentration of SDS is preferably 0.1% to 8%, more preferably 0.1% to 5%.

In one embodiment, the lysate may also contain RNase A, whose role is to remove RNA impurities from the system. The content of RNase A can be determined by those skilled in the art as needed, for example, 20 μg/ml.

In one embodiment, the final concentration of proteinase K is 0.005-4 mg/ml, preferably 0.005-2.5 mg/ml. The role of proteinase K is to degrade membrane proteins and proteins that bind to genomic DNA, thereby fully freeing genomic DNA. When the content of proteinase K is too low, for example, less than 0.005mg/ml, the protein combined with membrane protein and genomic DNA cannot be fully degraded, which may affect the subsequent application of genomic DNA, such as the BioNano optical spectrum labeling of genomic DNA; when proteinase K If the content is too high, for example, higher than 4mg/ml, excessive proteinase K will precipitate with genomic DNA, which will degrade the digestion system in subsequent applications, such as BioNano optical spectrum labeling that affects genomic DNA.

In one embodiment, proteinase K and lysate may be added sequentially or simultaneously.

The processing time and temperature of step a) above can be adjusted and determined by those skilled in the art according to experimental requirements. For example, it can be processed at a temperature of 30-65°C for 1-2 hours.

In one embodiment, the precipitating agent of the present invention comprises at least one inorganic salt and at least one alcohol. In another embodiment, the precipitating agent of the present invention consists of inorganic salts and alcohols. In the method of the present invention, examples of inorganic salts include, but are not limited to, ammonium acetate, sodium acetate, sodium chloride, lithium chloride, and the like; examples of alcohols include, but are not limited to, absolute ethanol, isopropanol, and the like. In the present invention, the concentration of inorganic salts suitable for precipitation of genomic DNA is 300-500 mM, preferably 350-450 mM, more preferably about 450 mM, and the concentration of alcohols is 50-80%, more preferably 60-70%, more preferably about 65 %.

The precipitation time of the above step b) can be adjusted and determined by those skilled in the art according to experimental requirements.

In one embodiment, the cleaning solution contains 70%-80% ethanol. In another embodiment, the cleaning solution consists of 70%-80% ethanol.

In one embodiment, the dissolving solution is selected from deionized water, Tris-HCl, TE buffer, and the like. Other known solutions that can be used to dissolve genomic DNA are also suitable for the present invention.

In one embodiment, the extracted genomic DNA is labeled with a dye in the presence of a labeling enzyme. Specifically, the labeling enzyme may be a commercially available or purified restriction enzyme or methyltransferase. The dye may be selected from BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanin, pyrene, and phthalocyanine. The conditions and methods for labeling the extracted genomic DNA can be adjusted by those skilled in the art according to actual needs. For example, in the presence of a labeling enzyme, the dye and genomic DNA can be incubated at a temperature suitable for the activity of the labeling enzyme for a sufficient time to allow the labeling reaction to occur sufficiently.

The purpose of purifying labeled genomic DNA is mainly to remove redundant or unreacted labeling enzymes and dyes in the labeling reaction. In one embodiment, the excess labeling enzyme is removed by high temperature inactivation or proteinase K digestion; and the excess dye is removed by precipitation or dialysis. Purification methods suitable for the present invention are known to those skilled in the art. For example, the labeled genomic DNA can be incubated with proteinase K for a period of time and then dialyzed through a filter to achieve purification.

In one embodiment, the purified genomic DNA backbone is stained with a dye to visualize the genomic DNA backbone. The dye used in this step may be, but not limited to, YOYO-1 or DAPI dye. Those skilled in the art can adjust the conditions and time of the dyeing step as needed.

In another embodiment, one or more of the above labeling, purification, and staining steps may be performed using commercially available kits, such as those provided by Bionano Corporation (eg, Bionano Prep DLS kit).

In one embodiment, the methods of the present invention for preparing single-molecule optical map labeled libraries are all performed in the liquid phase.

In the second aspect, the present invention also provides a kit for quickly preparing a single-molecule optical map labeling library, which contains a reagent for extracting a long fragment of genomic DNA, a labeling enzyme, at least two dyes, and optionally proteinase K.

In one embodiment, the reagent for extracting long fragments of genomic DNA further includes a lysis solution, proteinase K, optional RNase A, a precipitating agent, a washing solution, and a dissolution solution.

In one embodiment, the lysate contains NaCl, Tris-HCl (pH 7.4-8.0), EDTA (pH 8.0) and SDS. In another embodiment, the lysate consists of NaCl, Tris-HCl (pH 7.4-8.0), EDTA (pH 8.0) and SDS. The concentration of NaCl, Tris-HC (pH7.4-8.0) and EDTA (pH8.0) in the lysate can be adjusted and determined by routine experiments according to specific experimental requirements. For example, the concentration of NaCl is 0.1M-0.5M, Tris -The concentration of HCl (pH7.4-8.0) is 10mM-200mM, and the concentration of EDTA (pH8.0) is 10mM-100mM. In the present invention, the preferred SDS concentration is 0.1%-8%, more preferably 0.1-5%.

Preferably, the concentration of proteinase K used to extract long-length genomic DNA is 0.005-4 mg/ml, more preferably 0.005-2.5 mg/ml. When RNase A is present, its content is 20 μg/ml.

In one embodiment, the precipitating agent contains at least one inorganic salt and at least one alcohol. In another embodiment, the precipitating agent of the present invention consists of inorganic salts and alcohols. In the method of the present invention, examples of inorganic salts include, but are not limited to, ammonium acetate, sodium acetate, sodium chloride, and the like; examples of alcohols include, but are not limited to, absolute ethanol, isopropanol, and the like. The concentration of inorganic salts suitable for precipitation of genomic DNA in the present invention is 300-500 mM, preferably 350-450 mM, more preferably about 450 mM. The concentration of alcohol is 50-80%, more preferably 60-70%, more preferably about 65%.

In one embodiment, the cleaning solution contains 70%-80% ethanol. In another embodiment, the cleaning solution consists of 70%-80% ethanol.

In one embodiment, the dissolving solution is selected from deionized water, Tris-HCl, TE buffer, and the like.

In one embodiment, the labeling enzyme is a restriction nicking enzyme or a methyltransferase.

In one embodiment, examples of dyes include but are not limited to YOYO-1, BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanin, pyrene, phthalocyanine, and the like.

The method and kit according to the present invention can quickly prepare a single molecule optical spectrum labeling library in the liquid phase, which is suitable for platforms such as BioNano Genomics' Irys and Saphyr. The excellent technical effect of the present invention mainly depends on the following aspects:

(1) In terms of extracting long fragments of genomic DNA, all operations of the method of the present invention are completed in a single reaction tube, which does not involve the transfer of genomic DNA in different reaction tubes, thereby saving time and cost while avoiding Loss of genomic DNA and possible contamination. At the same time, the entire genomic DNA extraction process only includes four steps of lysis, precipitation, washing and dissolution, which takes 1-2 hours, greatly reducing the extraction time. In addition, the extraction method of the present invention is relatively safe, and will not harm the health of the operator. The reagents used in the extraction method of the present invention are safe and non-toxic, and at the same time avoid the use of highly toxic organic solvents such as phenol chloroform used in traditional genomic DNA precipitation methods. Finally, the length and quality of the extracted genomic DNA are reliable and highly repeatable. Compared with the traditional silica gel membrane column and magnetic bead purification method, the present invention avoids mechanical damage of genomic DNA. Compared with the classic extraction method based on low melting point agarose gel extraction, the present invention has significantly improved the operability and reproducibility of experiments.

(2) According to the method and kit of the present invention, the extracted long-length genomic DNA has good uniformity and a fixed total amount, and subsequent experiments can be carried out without measuring the concentration; the optical map labeling library thus obtained does not need to have a quantitative concentration Directly used in BioNano Genomics' Irys and Saphyr platforms.

(3) The entire process of preparing the optical map labeling library according to the method and kit of the present invention only takes about 8 hours, which greatly shortens the preparation time compared to the 3-4 days required by the traditional method.

The present invention will be further described below with reference to the drawings and embodiments. It should be understood that the following examples are merely illustrative and are not intended to limit the scope of the present invention.

BRIEF DESCRIPTION

Figure 1. Process for preparing a single-molecule optical map labeling library of long-length genomic DNA according to the method of the present invention.

Figure 2. Pulsed field electrophoresis results of the single-molecule optical map labeling library prepared according to Example 1.

Figure 3. Labeling results of long fragments of genomic DNA extracted with different proteinase K concentrations.

Figure 4. Labeling results of long genomic DNA extracted using different SDS concentrations.

Figure 5. Analysis results of chromosome structure variation in Example 3, showing the comparison results of the assembled two chromosomes with reference chromosome 5 and reference chromosome 14;

detailed description

Example 1. Preparation of a single-molecule optical map labeling library of long fragments of genomic DNA according to the method of the present invention

Step 1. Extract long genomic DNA according to the following steps

Take 0.3x10 ⁶ white blood cells, centrifuge at 2000g at room temperature for 2 minutes, and remove the supernatant. Add 30 μL of lysate (100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0), 0.50% SDS) to resuspend the cells, then add 2 μL of 2 mg/ml proteinase K to mix and place at 50°C Metal bath treatment for 1 hour. Then add 12 μL of 5M ammonium acetate and 90 μL of absolute ethanol. After inverting 10 times, place it on a shaker at room temperature and mix at 20 rpm for 5-10 minutes to allow the DNA to completely precipitate and form small clusters. The DNA was washed twice with 200 μL of 70% ethanol, and the DNA was dissolved with 42 μL of TE buffer (10 mmol/L Tris-HCl (pH 8.0); 1 mmol/L EDTA (pH 8.0)) to obtain long-length genomic DNA.

Step 2. Mark the extracted genomic DNA

Using BionanoPrep DLS Kit (Cat No.80005), prepare the marking mixture shown in Table 1 below in the dark conditions:

Table 1:

The labeling mixture was then incubated at 37°C for 2h and then flashed off.

Step 3. Purify labeled genomic DNA

Slowly add 5 μl of proteinase K to the labeling mixture obtained in step 2 to remove excess labeling enzyme DLE-1, and flash off the sample, then incubate at 50°C for 0.5 h, and flash off the sample again.

Then, the excess dye DL-Green is removed by dialysis. Specifically, add 25 μl 1×DLE1 buffer (5 μl 5×DLE1 buffer + 20 μl H2O from Bionano Prep DLS Kit) to the wells of a 24-well plate, and then place the filter on the buffer. After it is wet, add The genomic DNA of the excess labeled enzyme was removed, and the well was sealed. Leave at room temperature in the dark for 20 minutes. Repeat this dialysis step twice.

Step 4. Stain purified genomic DNA

Take 20μl of DNA from the genomic DNA obtained in step 3 into a 2ml round bottom dark tube, and add 40μl of pre-stained mixture (15μl 4×Flow, 12μl 5×DTT, 13μl nuclease-free H2O, all from Bionano Prep DLS Kit). After mixing, add YOYO-1 dye (3.2 μl dye per 300 ng of genomic DNA), rotate at room temperature for 2 hours, and then instantaneously separate the sample to obtain a single-molecule optical map labeling library of long-length genomic DNA.

Take 20μl of labeled library for pulse field electrophoresis to detect the size of DNA, the results are shown in Figure 2. It can be seen from FIG. 2 that the average length of genomic DNA in the single-molecule optical map marker library obtained by the method of the present invention is more than 200 kb, and the longest can reach Mb level.

Another 20μl labeled library was collected on BioNano's Saphyr platform to collect data. The results are shown in Table 2 below.

Table 2:

From the above results, it can be seen that the single-molecule optical map marker library prepared according to the present invention has good quality, meets the quality requirements of the marker, and can be used for genome assembly and further chromosome analysis.

Example 2. The effect of different concentrations of proteinase K and SDS on the labeling effect of optical map when extracting genomic DNA

In order to detect the effect of different concentrations of proteinase K and SDS cleavage on the optical spectrum labeling effect when extracting genomic DNA, according to step 1 of Example 1, different concentrations of proteinase K and SDS were used to extract long-length genomic DNA of leukocytes. Then, according to steps 2-4 of Example 1, a single-molecule optical map labeling library was prepared, and data collection was performed on the Saphyr platform of BioNano Genomics.

As shown in Figure 3, when the concentration of proteinase K is too high, for example, 5mg/ml, the labeling density and comparison rate of the extracted long DNA fragments are too low, and the proportion of false negative labels is too high to meet the ideal labeling requirements. (Determined according to the reference range). However, when the concentration of proteinase K is too low, for example, 0.003125 mg/ml, the labeling density and comparison rate of the extracted long DNA fragments are still too low, and the ideal labeling requirements cannot be achieved.

As shown in Figure 4, when the SDS concentration is too high, for example, 10%, the labeling density and comparison rate of the extracted long DNA fragments are extremely low, and the proportion of false negative labels is too high to meet the ideal labeling requirements (according to Reference range determination). However, when the SDS concentration is too low, for example, 0.05%, the labeling density and comparison rate of the extracted long DNA fragments are still too low, and the molecular length N50 is far beyond the reference range, which also fails to meet the ideal labeling requirements.

Therefore, in order to make the quality of the extracted long-length DNA high enough to meet the requirements of subsequent direct sequencing and assembly (for example, in the case of BioNano's single-molecule optical spectroscopy technology, the quality of the long-length DNA must be high enough to achieve the ideal The labeling requirements can be used for efficient genome assembly afterwards without affecting its completion and accuracy). When extracting long fragments of DNA, the concentration of proteinase K and SDS should be controlled within a certain range. For example, the protease concentration is preferably 0.005-4 mg/ml, more preferably 0.005-2.5 mg/ml; the SDS concentration is preferably 0.1-8%, more preferably 0.1-5%.

Example 3. Use of single-molecule optical map marker library prepared according to the present invention in chromosome structure variation analysis

In this embodiment, a blood sample with a balanced translocation is used, and the karyotype is 46,XX,t(5;14)(q11;q32) through karyotype analysis. Extract long genomic DNA from the blood sample according to the method described in Example 1 and prepare a single-molecule optical map marker library, then collect the data on the Saphyr platform, and use BioNanoSolve software to assemble the collected data to obtain N50 The genome map of 75.6Mb. Chromosome structural variation analysis of this genome map revealed that there were chromosome breaks in chr5:50217079-50228607 and chr14:90511140-90522191 (Figure 5), which is consistent with the results of karyotype analysis.

Therefore, the method and kit according to the present invention can quickly prepare a single-molecule optical map marker library, and can successfully perform genome assembly and chromosome structure variation analysis.

It should be noted that although some features of the present invention have been clarified through the above embodiments, they cannot be used to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Therefore, for those skilled in the art, several simple replacements can be made without departing from the concept and principle of the present invention, and these should be included in the protection scope of the present invention.

references

[1] Lam ET et al. Genome mapping on nanochannel arrays for structural variation analysis and sequence assembly. Nat Biotechnol. 2012 Aug; 30(8): 771-6.

[2] Barseghyan H.et.al.Next-generation mapping: a novel approach for detection of pathogenic structural variants with potential potential utility in clinical diagnostic.Genome Med. 2017 Oct 25; 9(1): 90.

[3] Grunwald et al. Reduced representation optical methylation mapping (R2OM2). BioRxiv, 2017 Mar 3.

[4]DaiYetetal.Single-moleculeopticalmappingenablesaccuratemoleculardiagnosisoffacioscapulohumeralmuseulardystrophy(FSHD)GenomemappingmononnanochannelnarraysforforstructuralvariationanalysisandBiosequence21.2018

[5] Chan EKF et al. Optical mapping reveals a higher level of genomic architecture architecture of chained fusions in cancer. Genome Res. 2018 May; 28(5): 726-738.

[6] Keeble-Gagnère G. Optical. Optical and physical mapping with local local finishing megabase-scale resolution of agronomically imported regions regions of the heat of Genome. Genome Biol. 2018 Aug 17; 19(1): 112.

Claims (26)

1. A method for quickly preparing a single-molecule optical map labeling library includes the following steps:

   (1) Extract long genomic DNA;

   (2) Mark the extracted genomic DNA;

   (3) Purification of labeled genomic DNA, and

   (4) Stain the skeleton of genomic DNA to obtain a library of single-molecule optical map markers.
2. The method of claim 1, wherein step (1) further comprises the following sub-steps:

   a) Add lysate and proteinase K to the sample to lyse the cells and release genomic DNA;

   b) Add a precipitant to the reaction system of step a) to obtain a precipitate of genomic DNA;

   c) Wash the resulting genomic DNA precipitate with a cleaning solution;

   d) Dissolve the genomic DNA in the dissolving solution.
3. The method of claim 1 or 2, wherein step (1) is performed in a single reaction tube.
4. The method of claim 2, wherein the lysate comprises NaCl, Tris-HCl, EDTA, and SDS.
5. The method of claim 4, wherein the concentration of the SDS is 0.1%-8%.
6. The method of claim 2, wherein the lysate comprises RNase A.
7. The method of claim 2, wherein the concentration of proteinase K is 0.005-4 mg/ml.
8. The method of claim 2, wherein the precipitating agent comprises at least one inorganic salt and at least one alcohol.
9. The method of claim 2, wherein the cleaning solution contains 70% ethanol.
10. The method of claim 2, wherein the dissolving solution is selected from deionized water, Tris-HCl, and TE buffer.
11. The method of claim 1, wherein step (2) includes incubating the dye with the genomic DNA extracted in step (1) in the presence of a labeling enzyme.
12. The method of claim 11, wherein the labeling enzyme is a restriction nicking enzyme or a methyltransferase.
13. The method of claim 11, wherein the dye is selected from BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanin, pyrene, and phthalocyanine.
14. The method of claim 1, wherein step (3) includes removing excess labeling enzyme by high temperature inactivation method or proteinase K digestion method; and removing excess dye by precipitation method or dialysis method.
15. The method of claim 1, wherein the dye used in step (4) is YOYO-1 or DAPI dye.
16. The method of claim 1, wherein the single-molecule optical map labeling library is suitable for the Irys or Saphyr platform of BioNano Genomics.
17. A kit for quickly preparing a single-molecule optical map labeling library, a reagent for extracting long fragments of genomic DNA, a labeling enzyme, at least two dyes, and optional proteinase K.
18. The kit according to claim 17, wherein the reagent for extracting long-length genomic DNA further comprises a lysis solution, proteinase K, optional RNase A, a precipitating agent, a cleaning solution, and a dissolution solution.
19. The kit of claim 18, wherein the lysate contains NaCl, Tris-HCl, EDTA, and SDS.
20. The kit of claim 19, wherein the concentration of the SDS is 0.1%-8%.
21. The kit of claim 18, wherein the concentration of proteinase K is 0.005-4 mg/ml.
22. The kit of claim 18, wherein the precipitant comprises at least one inorganic salt and at least one alcohol.
23. The kit of claim 18, wherein the cleaning solution contains 70% ethanol.
24. The kit of claim 18, wherein the dissolution solution is selected from deionized water, Tris-HCl, and TE buffer.
25. The kit of claim 17, wherein the labeling enzyme is a restriction nicking enzyme or a methyltransferase.
26. The kit of claim 17, wherein one dye is selected from BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanin, pyrene, and phthalocyanine, and the other dye is selected from YOYO-1 or DAPI dye .

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