CN111378647A

CN111378647A - Method and kit for rapidly preparing monomolecular optical spectrum marker library

Info

Publication number: CN111378647A
Application number: CN201811619579.4A
Authority: CN
Inventors: 毛爱平; 张海满; 张建光
Original assignee: Berry Genomics Co Ltd
Current assignee: Berry Genomics Co Ltd
Priority date: 2018-12-27
Filing date: 2018-12-27
Publication date: 2020-07-07
Also published as: WO2020133590A1

Abstract

The invention relates to a method for rapidly preparing a monomolecular optical spectrum marker library, which comprises the following steps: (1) extracting long fragment genome DNA; (2) labeling the extracted genomic DNA; (3) purifying the labeled genomic DNA, and (4) staining the backbone of the genomic DNA to obtain a library of single molecule optical map markers. The invention also relates to a kit for preparing the single-molecule optical map marker library.

Description

Method and kit for rapidly preparing monomolecular optical spectrum marker library

Technical Field

The invention relates to a method for rapidly preparing a single-molecule optical map marker library and a kit suitable for the method.

Background

Many diseases in humans are caused by structural variations in chromosomes. Examples of chromosome structure analysis methods that are commonly used at present include karyotype analysis, chip hybridization, and the like. However, these techniques have limitations such as low resolution, high cost, inability to cover the whole genome, etc. The development of long-fragment single-molecule optical mapping technology has made up the limitations of the above-mentioned technology. Irys and Saphyr systems from BioNano recognize DNA and label fluorescent DNA using restriction nicking or methyltransferase¹And then, the DNA single molecule is linearly expanded by utilizing the nano channel, and the ultralong single molecule high-resolution fluorescence imaging is carried out, so that a specific enzyme site distribution map can be generated. The monomolecular optical spectrum technology canDetection of chromosomal structural variation and genetic disease by assisting genome assembly². However, the preparation of the single-molecule optical map labeled library at the present stage has the disadvantages of long time consumption, complicated preparation process and the like, and is not suitable for clinical detection.

In addition, the optical mapping technique has an advantage in that the DNA can be labeled at a single molecule level, and thus the length of the genomic DNA fragment is one of the important factors determining the labeling effect of the optical mapping. It is generally required that the average length of the DNA fragments is more than 200Kb and the longest DNA fragments reach the Mb level. Currently, the low melting point agarose gel embedding-based solid phase method is generally adopted to obtain long-fragment genomic DNA for preparing an optical map marker library^3-6. The method can extract high-purity genome DNA up to Mb level, but comprises the steps of embedding cells into agarose gel blocks, carrying out proteinase K digestion treatment, melting the gel blocks, carrying out enzymolysis on the gel blocks, dialyzing the genome DNA and the like, and the operation process is very complicated and takes more than 24 hours. Moreover, the yield of the long-fragment genome DNA extracted by the method is often unstable and very sticky, and the concentration is difficult to measure due to poor uniformity, so that the quality of subsequent optical map markers is influenced. This solid phase long fragment genomic DNA extraction plus Direct Label Staining (DLS) by BioNano corporation made it a total of up to 3.5 days to prepare an optical map Labeling library. These factors are not favorable for large-scale popularization and application of the optical spectrum technology.

In order to solve the above problems, the present invention provides a method for rapidly preparing an optical mapping marker library based on a liquid phase system, which comprises four steps of extracting long-fragment genomic DNA, labeling the genomic DNA, purifying the labeled genomic DNA, and staining the genomic DNA. The method simplifies the flow of optical map marking, shortens the time from the original 3.5 days to 8 hours, and the finally obtained marking library has higher quality and strong repeatability, thereby being beneficial to the application of the single-molecule optical map technology in clinical detection.

Disclosure of Invention

The invention aims to solve the problems of long time consumption, complex process and the like of single-molecule optical spectrum library preparation at the present stage. The invention can realize the rapid preparation of the rapid monomolecular optical map marking library by extracting the long-fragment genome DNA through a liquid phase and simplifying the monomolecular optical map marking process.

Accordingly, in a first aspect, the present invention provides a method for rapidly preparing a library of single molecule optical map markers, comprising the steps of:

(1) extracting long fragment genome DNA;

(2) labeling the extracted genomic DNA;

(3) purifying the labeled genomic DNA, and

(4) and dyeing the skeleton of the genome DNA to obtain a single-molecule optical map marker library.

In one embodiment, the step of extracting the long-fragment genomic DNA comprises cell lysis, genomic DNA precipitation, genomic DNA washing, and genomic DNA lysis. In a preferred embodiment, the step of extracting the long fragment genomic DNA comprises the sub-steps of:

a) adding lysis solution and proteinase K to the sample to lyse the cells and release genomic DNA;

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

c) washing the resulting precipitate of genomic DNA with a washing solution;

d) the genomic DNA was dissolved in a dissolving solution.

In a preferred embodiment, the step of extracting the long-fragment genomic DNA consists of the above four substeps a) -d).

As used herein, "long-fragment genomic DNA" refers to genomic DNA having an average length of greater than 200Kb and up to the 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, and the like. Examples of bodily fluids include, but are not limited to, blood, serum, plasma, joint fluid, semen, urine, sweat, saliva, stool, cerebrospinal fluid, ascites, pleural fluid, bile, pancreatic fluid, and the like.

In one embodiment, the lysate comprises NaCl, Tris-HCl (pH7.4-8.0), EDTA (pH8.0), and SDS. In another embodiment, the lysate consists of NaCl, Tris-HCl (pH7.4-8.0), EDTA (pH8.0) and SDS. The concentration of NaCl, Tris-HCl (pH7.4-8.0) and EDTA (pH8.0) in the lysate may be adjusted and determined by routine experimentation according to the particular experimental requirements, e.g., concentration of NaCl from 0.1M to 0.5M, concentration of Tris-HCl (pH7.4-8.0) from 10mM to 200mM, concentration of EDTA (pH8.0) from 10mM to 100 mM. However, the concentration of SDS is critical to the quality of the extracted genomic DNA. Specifically, too low a concentration of SDS, for example, less than 0.1%, may result in insufficient cell lysis; if the concentration of SDS is too high, for example, more than 8%, an excessive amount of SDS may precipitate with the genomic DNA, which may affect the 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, which serves to remove RNA impurities from the system. The content of RNase A can be determined by those skilled in the art as required, for example, 20. mu.g/ml.

In one embodiment, the final concentration of proteinase K is 0.005-4mg/ml, preferably 0.005-2.5 mg/ml. Proteinase K acts to degrade membrane proteins and proteins bound to genomic DNA, thereby freeing the genomic DNA sufficiently. When the content of proteinase K is too low, for example, less than 0.005mg/ml, the membrane protein and the protein bound to the genomic DNA cannot be degraded sufficiently, and the subsequent application of the genomic DNA may be affected, for example, the BioNano optical map labeling of the genomic DNA is affected; when the content of proteinase K is too high, for example, higher than 4mg/ml, the excessive proteinase K will precipitate with the genomic DNA, and degrade the enzyme digestion system in the subsequent application, for example, the BioNano optical map marker affecting the genomic DNA.

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

The treatment time and temperature of step a) above can be adjusted and determined by the skilled person according to the experimental requirements. For example, the treatment may be carried out at a temperature of 30 to 65 ℃ for 1 to 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 an inorganic salt and an alcohol. In the method of the present invention, examples of the inorganic salt include, but are not limited to, ammonium acetate, sodium chloride, lithium chloride, and the like; examples of alcohols include, but are not limited to, absolute ethanol, isopropanol, and the like. The concentration of the inorganic salt suitable for precipitating genomic DNA in the present invention is 300-500mM, preferably 350-450mM, more preferably about 450mM, and the concentration of the alcohol is 50-80%, more preferably 60-70%, more preferably about 65%.

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

In one embodiment, the cleaning solution comprises 70% to 80% ethanol. In another embodiment, the cleaning solution consists of 70% to 80% ethanol.

In one embodiment, the lysis solution is selected from the group consisting of deionized water, Tris-HCl, TE buffer, and the like. Other known solutions that can be used to dissolve genomic DNA are also suitable for use in 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 endonuclease or a methyltransferase. The dye may be selected from BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanins, pyrene and phthalocyanines. The conditions and methods for labeling the extracted genomic DNA can be adjusted by those skilled in the art according to the actual need. For example, the dye may be incubated with the genomic DNA in the presence of the labeling enzyme at a temperature suitable for the activity of the labeling enzyme for a time sufficient for the labeling reaction to occur.

The purpose of purifying the labeled genomic DNA is mainly to remove excess or unreacted labeling enzyme and dye in the labeling reaction. In one embodiment, excess labeled enzyme is removed by high temperature inactivation or proteinase K digestion; and removing excess dye by precipitation or dialysis. Purification methods suitable for use in the present invention are known to those skilled in the art. For example, the labeled genomic DNA may 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 backbone of the genomic DNA. The dye used for this step may be, but is not limited to, YOYO-1 or DAPI dye. The conditions and time of this dyeing step can be adjusted as desired by the skilled person.

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

In one embodiment, the methods of the invention for preparing a library of single molecule optical map markers are all performed in liquid phase.

In a second aspect, the present invention also provides a kit for rapidly preparing a single-molecule optical map marker library, comprising reagents for extracting long-fragment genomic DNA, a marker enzyme, at least two dyes, and optionally proteinase K.

In one embodiment, the reagent for extracting long-fragment genomic DNA further comprises a lysis solution, proteinase K, optionally RNase a, a precipitating agent, a washing solution, and a lysis solution.

In one embodiment, the lysate comprises NaCl, Tris-HCl (pH7.4-8.0), EDTA (pH8.0), and SDS. In another embodiment, the lysate consists of NaCl, Tris-HCl (pH7.4-8.0), EDTA (pH8.0) 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 experimentation according to the particular experimental requirements, e.g., concentration of NaCl from 0.1M to 0.5M, concentration of Tris-HCl (pH7.4-8.0) from 1OmM to 200mM, and concentration of EDTA (pH8.0) from 10mM to 100 mM. In the present invention, the concentration of SDS is preferably 0.1% to 8%, more preferably 0.1 to 5%.

Preferably, the concentration of proteinase K used for extraction of long-fragment genomic DNA is 0.005-4mg/ml, more preferably 0.005-2.5 mg/ml. When RNase A is present, it is present in an amount of 20. mu.g/ml.

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

In one embodiment, the lysis solution is selected from the group consisting of deionized water, Tris-HCl, TE buffer, and the like.

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

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

The method and the kit according to the invention can rapidly prepare a single-molecule optical mapping mark library in a liquid phase, and are suitable for platforms such as Irys and Saphyr of BioNano Genomics. The excellent technical effect of the invention mainly depends on the following aspects:

(1) in the aspect of extracting long-fragment genomic DNA, all operations of the method are completed in a single reaction tube, and the transfer of the genomic DNA in different reaction tubes is not involved, so that the loss and possible pollution of the genomic DNA are avoided while the time and the cost are saved. Meanwhile, the whole extraction process of the genome DNA only comprises four steps of cracking, precipitating, cleaning and dissolving, the time is 1-2 hours, and the extraction time is greatly shortened. In addition, the extraction method of the invention is safe and does not cause harm to the health of operators. The reagents used in the extraction method of the invention are safe and nontoxic, and the use of highly toxic organic solvents such as phenol chloroform used in the traditional genomic DNA precipitation method is avoided. Finally, the extracted genome DNA has reliable length and quality and strong repeatability. Compared with the traditional silica gel membrane column and magnetic bead purification method, the invention avoids the mechanical damage of the genome DNA. Compared with the classical method for extracting long-fragment genome DNA based on low-melting-point agarose gel embedding, the method provided by the invention has the advantages that the operability and repeatability of the experiment are obviously improved.

(2) According to the method and the kit, the extracted long-fragment genome DNA has good uniformity and fixed total amount, and subsequent experiments can be carried out without measuring concentration; the optical mapping library thus obtained was used directly without quantitative concentration for Irys and Saphyr platforms from BioNano Genomics.

(3) The whole process for preparing the optical map marker library by using the method and the kit only needs about 8 hours, and the preparation time is greatly shortened compared with the 3-4 days required by the traditional method.

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

Drawings

FIG. 1 is a flow chart of a single-molecule optical map marker library for preparing long-fragment genomic DNA according to the method of the present invention.

FIG. 2 shows the results of pulsed field electrophoresis of the single-molecule optical spectrum marker library prepared according to example 1.

FIG. 3. labeling results of long-fragment genomic DNA extracted using different proteinase K concentrations.

FIG. 4. labeling results of long-fragment genomic DNA extracted using different SDS concentrations.

FIG. 5 results of structural variation analysis of chromosomes according to example 3, showing the alignment of two chromosomes assembled with

reference chromosomes

5 and 14.

Detailed Description

Example 1 preparation of Single-molecule optical map marker library of Long-fragment genomic DNA according to the method of the present invention

Step 1. extraction of Long fragment genomic DNA according to the following procedure

Take 0.3x10⁶The white blood cells were centrifuged at 2000g for 2 minutes at room temperature, and the supernatant was removed. mu.L of lysis buffer (100mM NaCl, 10mM Tris-HCl (pH8.0), 25mM EDTA (pH8.0), 0.50% SDS) was added to resuspend the cells, 2. mu.L of proteinase K (2 mg/ml) was added thereto, mixed well, and placed in a metal bath at 50 ℃ for 1 hour. Then 12. mu.L of 5M ammonium acetate and 90. mu.L of absolute ethanol were added, and after inverting 10 times, the mixture was slowly mixed for 5 to 10 minutes at room temperature and 20rpm in a shaker to completely precipitate the DNA and form a pellet. The DNA was washed 2 times with 200. mu.L of 70% ethanol, and the DNA was dissolved in 42. mu.L of TE buffer (10mmol/L Tris-HCl (pH 8.0); 1mmol/L LEDTA (pH 8.0)) to obtain a long-fragment genomic DNA.

Step 2. marking the extracted genomic DNA

Using the Bionano Prep DLS Kit (Cat No.80005), labeling mixtures as shown in Table 1 below were prepared under exclusion of light:

table 1:

the labeling mixture was then incubated at 37 ℃ for 2h and then instantaneously detached.

Step 3. purification of the labeled genomic DNA

Mu.l proteinase K was slowly added to the labeling mixture obtained in step 2 to remove the excess labeling enzyme DLE-1, and the sample was transiently detached, followed by incubation at 50 ℃ for 0.5h, and again transiently detached.

Then, excess dye DL-green was removed by dialysis, specifically, 25 μ l of 1 × DLE1 buffer (5 μ l of 5 × DLE1 buffer +20 μ l H2O from Bionano Prep DLs Kit) was added to the wells of a 24-well plate, then the filters were placed on the buffer, after it was wetted, genomic DNA with excess labeling enzyme removed was added and the wells were sealed, placed in the dark at room temperature for 20 minutes.

Step 4 staining purified genomic DNA

Mu.l of DNA was taken from the genomic DNA obtained in step 3 and placed in a 2ml round bottom Buffer tube and 40. mu.l of a prestained mixture (15. mu.l of 4 × Flow Buffer, 12. mu.l of 5 × DTT, 13. mu.l of nuclease-free H2O, all from BioNanoPrep DLS Kit) was added after mixing, YOYO-1 dye (3.2. mu.l dye per 300ng genomic DNA) was added, spun at room temperature for 2 hours and then the sample was isolated instantaneously to obtain a single molecule optical map labeling library of long-fragment genomic DNA.

20 μ l of the labeled library was subjected to pulsed field electrophoresis to examine the DNA size, and the results are shown in FIG. 2. As can be seen from FIG. 2, the average length of the genomic DNA in the single-molecule optical map marker library obtained by the method of the present invention is 200kb or more, and the maximum length can reach Mb level.

Another 20. mu.l of the labeled library was collected on a Saphyr platform from BioNano, as shown in Table 2 below.

Table 2:

from the above results, the single-molecule optical map marker library prepared according to the invention has good quality, meets the quality requirement of the marker, and can be used for genome assembly and further chromosome analysis.

Example 2 Effect of different concentrations of proteinase K and SDS on the labeling Effect of optical profiles in extracting genomic DNA

In order to examine the effect of proteinase K and SDS lysis at different concentrations on the optical mapping labeling effect when extracting genomic DNA, long-fragment genomic DNA of leukocytes was extracted using proteinase K and SDS at different concentrations according to step 1 of example 1. A single molecule optical map marker library was then prepared according to steps 2-4 of example 1 and data collection was performed on the Saphyr platform of the BioNano Genomics company.

As shown in FIG. 3, when the concentration of proteinase K is too high, for example, 5mg/ml, the labeling density and the alignment rate of the extracted long DNA fragments are too low, and the proportion of false negative markers is too high to meet the ideal labeling requirement (determined by the reference range). When the concentration of proteinase K is too low, for example 0.003125mg/ml, the labeling density and alignment ratio of the extracted long-fragment DNA are still too low to meet the ideal labeling requirement.

As shown in FIG. 4, when the concentration of SDS is too high, for example, 10%, the labeling density and the alignment rate of the extracted long-fragment DNA are very low, and the proportion of false negative markers is too high to meet the ideal labeling requirement (determined by the reference range). When the concentration of SDS is too low, for example 0.05%, the labeling density and alignment ratio of the extracted long-fragment DNA are still too low, and the molecular length N50 is far beyond the reference range, which also fails to meet the ideal labeling requirement.

Therefore, in order to make the quality of the extracted long-fragment DNA high enough to satisfy the requirement of subsequent direct sequencing and assembly (for example, in the case of the single-molecule optical mapping technology of BioNano, the quality of the long-fragment DNA is high enough to reach the ideal labeling requirement, and then the genome assembly can be effectively carried out without affecting the completeness and accuracy), the concentration of proteinase K and the concentration of SDS should be controlled within a certain range when the long-fragment DNA is extracted. For example, the protease concentration is preferably 0.005-4mg/ml, more preferably 0.005-2.5 mg/ml; the concentration of SDS is preferably 0.1 to 8%, more preferably 0.1 to 5%.

Example 3 use of Single molecule optical map marker libraries prepared according to the invention in analysis of chromosomal structural variation

This example uses a blood sample with a balanced translocation with a karyotype of 46, XX, t (5; 14) (q 11; q32) by karyotype analysis. From this blood sample, long-fragment genomic DNA was extracted and a single-molecule optical map marker library was prepared according to the method described in example 1, and then data was collected on a Saphyr platform and genome-assembled using BioNano Solve software to obtain a genome map of 75.6Mb N50. The genomic map was subjected to a chromosomal structural variation analysis and found to be similar in chr 5: 50217079 50228607 and chr 14: 90511140 and 90522191 have chromosome breaks (FIG. 5), which are consistent with the karyotype analysis results.

Therefore, the method and the kit according to the present invention can rapidly prepare a single-molecule optical map marker library and can successfully perform genome assembly and structural variation analysis of chromosomes.

It should be noted that, although some features of the present invention have been illustrated by the above embodiments, the present invention is not limited thereto. Various modifications and alterations to this invention will become apparent to those skilled in the art. It will thus be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims.

Reference to the literature

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

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

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

[4]Dai Y et al.Single-molecule optical mapping enables accuratemolecular diagnosis of facioscapulohumeral muscular dystrophy(FSHD)Genomemapping on nanochannel arrays for structural variation analysis and sequenceassembly.BioRxiv，2018Mar 21.

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

[6]Keeble-Gagnère G et al.Optical and physical mapping with localfinishing enables megabase-scale resolution of agronomically importantregions in the wheat genome.Genome Biol.2018Aug 17；19(1)：112.

Claims

1. A method for rapidly preparing a single-molecule optical map marker library comprises the following steps:

(1) extracting long fragment genome DNA;

(2) labeling the extracted genomic DNA;

(3) purifying the labeled genomic DNA, and

2. The method of claim 1, wherein step (1) further comprises the sub-steps of:

c) washing the resulting precipitate of genomic DNA with a washing solution;

d) the genomic DNA was dissolved in a dissolving solution.

3. The process of claim 1 or 2, wherein step (1) is carried out in a single reaction tube.

4. The method of claim 2, wherein the lysis solution comprises NaCl, Tris-HCl, EDTA, and SDS.

5. The method of claim 4, wherein the concentration of SDS is 0.1% -8%.

6. The method of claim 2, wherein the lysis solution 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 wash solution comprises 70% ethanol.

10. The method of claim 2, wherein the lysis solution is selected from the group consisting of deionized water, Tris-HCl, and TE buffer.

11. The method of claim 1, wherein step (2) comprises 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 nickase or a methyltransferase.

13. The method of claim 11, wherein the dye is selected from the group consisting of BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanins, pyrene, and phthalocyanine.

14. The method of claim 1, wherein the step (3) comprises removing the excess labeled enzyme by a high temperature inactivation method or a proteinase K digestion method; and removing excess dye by precipitation or dialysis.

15. The method according to claim 1, wherein the dye used in step (4) is YOYO-1 or DAPI dye.

16. The method of claim 1, wherein the library of single molecule optical map markers is suitable for use with the Irys or Saphyr platforms of the company bionanogomoics.

17. A kit for rapidly preparing a single-molecule optical map marker library is used for extracting a reagent of long-fragment genomic DNA, a marker enzyme, at least two dyes and optional proteinase K.

18. The kit of claim 17, wherein the reagents for extracting long-fragment genomic DNA further comprise lysis buffer, proteinase K, optionally RNase a, precipitation reagent, wash solution and lysis solution.

19. The kit of claim 18, wherein the lysis solution comprises NaCl, Tris-HCl, EDTA, and SDS.

20. The kit of claim 19, wherein the concentration of SDS is between 0.1% and 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 precipitating agent comprises at least one inorganic salt and at least one alcohol.

23. The kit of claim 18, wherein the wash solution comprises 70% ethanol.

24. The kit of claim 18, wherein the lysis solution is selected from the group consisting of deionized water, Tris-HCl, and TE buffer.

25. The kit of claim 17, wherein the labeling enzyme is a restriction nickase 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.