CN108265049B - Whole genome interaction library and construction method thereof - Google Patents
Whole genome interaction library and construction method thereof Download PDFInfo
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Abstract
The invention provides a whole genome interaction library and a construction method thereof. The construction method comprises the following steps: step S1, connecting the ends of the DNA-protein cross-linked compound generated by enzyme digestion by using a primer sequence to form a ring-shaped compound; step S2, performing decrosslinking on the annular compound to obtain annular DNA; and step S3, carrying out fragmentation library construction on the circular DNA to obtain a whole genome interaction library. The method uses a segment of exogenous known DNA sequence to connect the tail ends of the segments of the enzyme digestion products, so that a circular compound is formed by the DNA-protein cross-linked compound, then the protein in the circular compound is removed through grafting and cross-linking, so that circular DNA is obtained, and the exogenous known DNA sequence carried in the circular DNA is used for constructing a fragmentation library, so that a whole genome interaction library for the whole genome interaction research can be obtained, and the method for researching the whole genome interaction is simple and convenient and has wide operability.
Description
Technical Field
The invention relates to the field of high-throughput sequencing library construction, in particular to a whole genome interaction library and a construction method thereof.
Background
Early studies on regulation of gene expression were mostly based on that the DNA strand is a linear strand, i.e., it is thought that promoters, exons, introns, enhancers, insulators, etc. are linearly distributed on the DNA strand. In fact, eukaryotic DNA is stored in the nucleus in a highly folded, concentrated chromatin fashion. Chromatin has spatially higher order structure and conformation; moreover, the higher structure and conformation play a very important role in the process of gene transcription and regulation. Therefore, to completely analyze the processes of gene transcription, DNA replication, DNA repair, etc., it is necessary to understand the spatial organization of chromatin, such as the spatial distribution of genome, the mutual contact and aggregation mode, the distribution of regulatory elements, the structural dynamics of chromatin, etc., which will make our understanding of the most complex behavior of life.
Currently, many molecular experimental techniques are used to study the three-dimensional structure and interactions of chromatin, such as fluorescence-labeled in situ hybridization (FISH) technique can be used to study the three-dimensional structure of intracellular chromatin (Yokoteatal, 1995), but its resolution is limited and the interaction of a large range of discrete gene loci in the genome cannot be well studied.
Chromatin conformation capture (3C) techniques were originally used to study the spatial conformation of chromatin upon yeast gene expression (dekkeretal, 2002), and were later also used to study the interactions between higher mammalian chromatin (skoketal, 2007). The principle of chromatin conformation capture (3C) is: as shown in fig. 1, (1) fixation of interacting chromatin sites within the nucleus with formaldehyde; (2) cutting the DNA into segments by using restriction enzymes; (3) ligating the ends of the fragments with a DNA ligase to capture a library containing the contacted DNA fragments (i.e., a 3C library); (4) detecting DNA fragment connection sites of the library by using a PCR (polymerase chain reaction) or sequencing method to obtain the frequency of mutual contact of different sites of chromatin; (5) and finally, carrying out data analysis to deduce the spatial position information of the chromatin, thereby obtaining a map of the chromatin interaction sites.
Experimental analysis based on 3C techniques is only a study focused on the interaction of one-to-one sites in the genome (dekkeretal, 2002). Later, on the basis of the 3C technical principle, a 4C (circular chromatin transformation capture, or chromatographic transformation capture-on chip) technology (Simoniset, 2006; Zhaoetal, 2006) is developed, namely a circular chromatin conformation capture technology or a chip chromatin conformation capture technology, wherein the principle of the 4C technology is that in the 3C principle analysis process, DNA fragments can be connected through ligase to form circular connection, then reverse PCR (polymerase chain reaction) is carried out, and finally the product is subjected to sequence analysis, so that the interaction research and analysis of one-to-many sites in chromatin can be obtained.
5C (chromosome conformation capture copy) technology (Dostimetal, 2006), also called 3C carbon copy technology, which increases the detection flux of 3C technology on interaction sites by amplifying ligation mediated products, thereby realizing the interaction research analysis of multiple sites by multiple sites. As can be seen, these techniques only allow the study of the interaction of some sites in chromatin, but do not allow the unbiased analysis of the interaction between all sites throughout the genome.
In 2009, a High-throughput/resolution chromosome conformation capture, which was developed by Lieberman-Aiden et al (2009) based on the 3C technical principle, is an experimental technique for studying genome-wide three-dimensional conformations and analyzing the interaction thereof. The Hi-C is more and more widely applied due to the fact that the Hi-C is based on a high-throughput sequencing technology and researches and analyzes the characteristics of interaction among all sites of a genome. Hi-C can be used for researching the interaction relation among all chromatin, and analyzing the problems of gene regulation and expression, for example, Lu and the like (2013) utilize Hi-C data analysis and prediction of the regulation and control relation of the far-end regulation and control elements such as an enhancer, an insulator, a suppressor and the like in a gene transcription and control region; babaei et al (2015) predicted a co-expression model for murine gene regulation by Hi-C technical studies. The principle of the Hi-C technique is as follows: (1) formaldehyde crosslinking: firstly, fixing DNA-protein or protein-protein compound which is crosslinked together or closely spaced in a natural state in cells by formaldehyde; (2) digestion and end labeling: digesting and separating chromatin with a restriction enzyme and labeling fragment ends with biotin; (3) fragment end ligation: connecting different cut ends by using DNA ligase to form a circular chimeric molecule; (4) purification and shearing: purifying the circular molecules, shearing and crushing the circular molecules into DNA fragments; (5) establishing a Hi-C library: using a biotin labeling precipitation technology to obtain a labeled target DNA fragment, selecting a DNA fragment with a proper size, and establishing a Hi-C library (Hi-C library); (6) sequencing analysis: high throughput sequencing techniques were used to double-end sequence (paired-end sequencing) the Hi-C library containing samples, resulting in a large number of paired-end fragments (paired-end reads) for analysis. There are now a very large number of Hi-C datasets on NCBI, covering a variety of cell lines of different species (Lieberman-aidenet al, 2009; nanoganeet al, 2013; mizuguchiet al, 2014; raoet al, 2014; lietal, 2015 a; maeal, 2015; sahlentatal, 2015).
In the genome interaction research method based on the method, time is consumed, and the repeatability is not very stable so far; the cost is high; the low-frequency gene interaction type detection efficiency is low; the operation steps are complicated, different enzymes are involved, and the equipment is numerous; and biotin labeling precipitation is needed to obtain a Hi-C library so as to carry out high-throughput sequencing and the like. Therefore, a new high-throughput genome interaction identification method is needed, which simplifies the experimental steps.
Disclosure of Invention
The invention mainly aims to provide a whole genome interaction library and a construction method thereof, and aims to solve the problems of complexity and long time consumption of the library construction method in the prior art.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method for constructing a genome-wide interaction library, the method comprising: step S1, connecting the ends of the DNA-protein cross-linked compound generated by enzyme digestion by using a primer sequence to form a ring-shaped compound; step S2, performing decrosslinking on the annular compound to obtain annular DNA; and step S3, carrying out fragmentation library construction on the circular DNA to obtain a whole genome interaction library.
Further, step S1 includes: step S11, performing first enzyme digestion treatment on the DNA-protein cross-linked compound by using a first endonuclease to obtain a first enzyme digestion product; and step S12, annealing and connecting the first enzyme digestion product and the primer sequence to obtain a circular compound.
Further, step S3 includes: step S31, performing secondary enzyme digestion treatment on the circular DNA by adopting second endonuclease treatment to obtain a second enzyme digestion product; step S32, carrying out cyclization treatment on the second enzyme digestion product to obtain a cyclization product; and step S33, performing reverse PCR on the cyclization product by using the primer sequence to obtain a whole genome interaction library.
Furthermore, the primer sequence is a sequence with the restriction enzyme sticky ends at two ends, the restriction enzyme sticky ends are the same as the sticky ends generated by the restriction enzyme of the first endonuclease, and the primer sequence does not contain the restriction enzyme cutting sites of the second endonuclease.
Further, the length of the primer sequence is 10-40 bp.
Further, step S33 includes: designing two amplification primers in opposite directions on the primer sequence; and carrying out reverse PCR on the cyclization product by using the amplification primer to obtain a whole genome interaction library.
Further, before performing step S1, the construction method further includes a step of obtaining a DNA-protein cross-linked complex, the step of obtaining the DNA-protein cross-linked complex including: fixing a cell sample to be detected by using formaldehyde to obtain fixed cells; and (3) cracking the fixed cells to obtain the DNA-protein cross-linked complex.
According to a second aspect of the present invention, there is provided a genome-wide interaction library constructed by any one of the above-described construction methods.
According to a third aspect of the present invention, there is provided a whole genome interaction library, comprising a target fragment and a foreign sequence linked to the target fragment, the foreign sequence comprising a sequence of a cleaved cohesive end of an endonuclease that cleaves a DNA-protein cross-linked complex when the whole genome interaction library is constructed.
By applying the technical scheme of the invention, biotin does not need to mark the tail ends of the enzyme digestion product fragments, but a segment of exogenous known DNA sequence is used for connecting the tail ends of the enzyme digestion product fragments, so that the DNA-protein cross-linked compound forms a circular compound, then the protein in the circular compound is removed through cross-linking, so that circular DNA is obtained, and the exogenous known DNA sequence carried in the circular DNA is utilized for constructing a fragmentation library, so that the whole genome interaction library for whole genome interaction research can be obtained. The application provides a simple and convenient method for researching whole genome interaction with wide operability on the basis of the existing Hi-C library construction method.
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The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows a schematic flow chart of a method for constructing a chromatin conformation capture library in the prior art; and
FIG. 2 is a detailed flow chart of the construction method of the genome-wide interaction library according to a preferred embodiment of the present application.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
As mentioned in the background, the Hi-C library construction method in the prior art has a problem of tedious and long time-consuming steps, and in order to improve the situation, the inventors have conducted intensive studies on the Hi-C library construction method, and in an exemplary embodiment of the present application, a method for constructing a genome-wide interaction library is provided, as shown in fig. 2, the method comprising: step S1, connecting the ends of the DNA-protein cross-linked compound generated by enzyme digestion by using a primer sequence to form a ring-shaped compound; step S2, performing decrosslinking on the annular compound to obtain annular DNA; and step S3, carrying out fragmentation library construction on the circular DNA to obtain a whole genome interaction library.
According to the file construction method provided by the application, biotin is not needed to mark the tail ends of the enzyme digestion product fragments, a section of exogenous known DNA sequence is used for connecting the tail ends of the enzyme digestion product fragments, the DNA-protein cross-linked compound forms a circular compound, then protein in the circular compound is removed through connecting and cross-linking, so that circular DNA is obtained, the exogenous known DNA sequence carried in the circular DNA is used for constructing a fragmentation library, and the whole genome interaction library for whole genome interaction research can be obtained. The application provides a simple and convenient method for researching whole genome interaction with wide operability on the basis of the existing Hi-C library construction method.
The step of ligating the cleaved fragments in step S1 described above first includes a step of generating cleaved fragments. Therefore, in a preferred embodiment of the present application, as shown in fig. 2, the step S1 includes: step S11, performing first enzyme digestion treatment on the DNA-protein cross-linked compound by using a first endonuclease to obtain a first enzyme digestion product; and step S12, annealing and connecting the first enzyme cutting product and the primer sequence to obtain a circular compound.
In step S3, the operation of constructing the fragmentation library of the circular DNA may be improved based on the existing operation of constructing the circular DNA library. In a preferred embodiment, as shown in fig. 2, the step S3 includes: step S31, performing secondary enzyme digestion treatment on the circular DNA by adopting second endonuclease treatment to obtain a second enzyme digestion product; step S32, carrying out cyclization treatment on the second enzyme digestion product to obtain a cyclization product; and step S33, reverse PCR is carried out on the cyclization product by utilizing the primer sequence to obtain a whole genome interaction library.
In another preferred embodiment of the present application, as shown in fig. 2, the step S33 includes: designing two amplification primers in opposite directions on the primer sequence; performing reverse PCR on the cyclization product by using an amplification primer to obtain a whole genome interaction library
Aiming at the measure of obtaining the target fragment by biotin precipitation in the existing Hi-C library construction method, on the basis of utilizing the exogenous known DNA sequence to link the ends of the fragments of the enzyme digestion product, the skilled person can fully utilize the exogenous known DNA sequence to purify and fragment the target fragment for library construction, for example, by designing two reverse primers on the known DNA sequence to perform reverse PCR on a mixed fragment, on one hand, the target fragment carrying only the exogenous known DNA sequence can be obtained, and on the other hand, the target fragment can also be amplified for deep sequencing.
In the library construction method, the steps of connecting the ends of the enzyme digestion product fragments by using the primer sequences are various, and the steps can be reasonably selected according to the structural characteristics of the primer sequences. For example, if the primer sequence is a sequence with a sticky end, the sticky end of the primer sequence can be designed to be the same as the sticky end generated by the enzyme digestion of the DNA-protein cross-linked complex, so that the primer sequence which is the same as the sticky end of the enzyme digestion fragment can be used for realizing the connection of adjacent enzyme digestion fragments only by automatic sticky end connection without the action of additional enzyme. Thus, in a preferred embodiment of the present application, the primer sequence is a sequence with cohesive ends at both ends, the cohesive ends are the same as those generated by the first endonuclease, and the primer sequence does not include the cleavage site of the second endonuclease. The primer sequence does not contain the enzyme cutting site of the second endonuclease, so that the exogenous DNA sequence is prevented from being cut off in the second enzyme cutting, and the exogenous sequence cannot be effectively utilized to screen and purify a target fragment.
The length of the primer sequence is not particularly required, as long as the function of connecting the enzyme-digested fragments can be realized. In order to further increase the length of the target fragment in the constructed library, in a preferred embodiment, the length of the primer sequence is 10-40 bp. The length range can realize the efficient connection of the tail ends of the enzyme digestion fragments, reduce the operation steps of biotin labeling, purification and precipitation, and obtain a full-gene interaction library with richer interaction sites and more comprehensive interaction sites by a simple PCR method, and the proportion of non-target fragments in the library is smaller.
In the library construction method, the step of obtaining the DNA-protein cross-linked complex may be performed by a conventional method. In a preferred embodiment, before performing step S1, the construction method further includes a step of obtaining a DNA-protein cross-linked complex, the step of obtaining the DNA-protein cross-linked complex including: fixing a cell sample to be detected by using formaldehyde to obtain fixed cells; and (3) cracking the fixed cells to obtain the DNA-protein cross-linked complex. In the step of fixing the cells by formaldehyde, formaldehyde with a final concentration of 1 wt% is fixed. And (3) a step of cracking the fixed cells, namely cracking the cells by adopting a conventional cell lysate to obtain a cracking product, and then extracting the DNA-protein cross-linked complex from the cracking product. The specific cracking and extraction operation can be realized by adopting the existing operation steps.
In a second exemplary embodiment of the present application, a genome-wide interaction library is provided, which is constructed by any one of the above-described construction methods. The whole genome interaction library constructed by the construction method has the advantages of richer and more comprehensive interaction sites.
In a third exemplary embodiment of the present application, a genome-wide interaction library is provided, which comprises a target fragment and a foreign sequence linked to the target fragment, wherein the foreign sequence comprises a sequence of a cleaved cohesive end of an endonuclease that cleaves a DNA-protein cross-linked complex when the genome-wide interaction library is constructed. The whole genome interaction library has the advantages of richer and more comprehensive interaction sites.
The advantageous effects of the present application will be further described with reference to specific examples.
In the following examples and comparative examples, the endonuclease-related reagents used were those derived from NEB, and the remaining reagents were commercially available products, if specifically noted.
Example 1
2g of fresh leaves (about 25X 10) grown for three weeks were selected6The cells) as a starting material, and adopting formaldehyde with the mass concentration of 1% to crosslink for 40-60 min at room temperature to obtain cells with fixed chromatin, wherein the specific library construction steps are as follows:
1) the cells are lysed. Each cell (25X 10)6One) 1ml of iced lysis buffer (10mM Tris-HCl,30mM NaCl, 0.2% NP-40, 10% protease inhibitor, pH7.4) was added and placed on ice for 15 min. The cells were flooded with 4 groups of 30 cells each on ice using a tissue homogenizer. The lysate was centrifuged at 2,000Xg for 5min at room temperature. The samples were washed twice with pre-cooled restriction enzyme-corresponding digestion buffer (1 XNEBuffer Cutsmart) and collected by centrifugation at 2000Xg for 5 min. Use of260 μ L of the digestion buffer resuspended the sample.
2) Chromatin digestion. Adding 1560 μ L of restriction enzyme buffer (1 XNEBuffer Cutsmart) corresponding to restriction enzyme and 190 μ L of 2% SDS into the sample treated in the previous step, slightly inverting and mixing, and incubating at 65 deg.C for 10 min; add 220. mu.L of 20% Triton-X-100 to quench the SDS; 2000U of restriction enzyme HindIII (enzyme concentration should be lower to avoid star activity) was added to each tube; digestion was carried out overnight in a shaker at 37 ℃. The endonuclease was inactivated by incubation at 65 ℃ for 20 min.
3) Introduction of primer annealing product. Each tube was added with the primer annealing product designed in advance and T4Ligation buffer at a final concentration of 1X and T4DNA ligase at 200U, and ligated overnight at 16 ℃.
4) De-crosslinking and DNA extraction. 200 μ L of 10mg/ml protein kinase K was added and incubated at 65 ℃ for about 4h to remove cross-linking. The reconnected DNA fragment was extracted using phenol-chloroform and dissolved in 100. mu.L of 1 XTE buffer, Ph8.0.
5) Resuspended DNA was subjected to 30kDa ultrafilter to remove salt and DTT. The purified DNA was added with 2. mu.L of RNAase A (1mg/ml) and incubated at 37 ℃ for 30min to digest the RNA.
6) The product from the previous step was digested with the other restriction enzyme MspI overnight at 37 ℃. The digested DNA fragment was extracted by phenol-chloroform method, dissolved in 1 XTE buffer (100. mu.L), and diluted. The diluted product was added to a T4ligation buffer at a final concentration of 10X and 200U of T4DNA ligase, and ligated overnight at 16 ℃ to circularize itself.
7) Small fragment library construction
The two interactive DNAs were amplified by inverse PCR, and the inverse PCR amplification product was subjected to a second amplification for high throughput sequencing.
Blast comparison was performed on the high-throughput sequencing results to find the number of interacting DNA sites, and the specific analysis results are shown in table 1.
Table 1:
| Chr01 | Chr02 | Chr03 | Chr04 | Chr05 | Chr06 | Chr07 | Chr08 | Chr09 | Chr10 | Chr11 | Chr12 | |
| Chr01 | 1231 | 450 | 342 | 432 | 356 | 364 | 234 | 457 | 796 | 421 | 456 | 321 |
| Chr02 | 450 | 1453 | 456 | 324 | 453 | 456 | 543 | 247 | 467 | 564 | 865 | 345 |
| Chr03 | 342 | 456 | 1873 | 345 | 321 | 135 | 157 | 145 | 187 | 243 | 331 | 254 |
| Chr04 | 432 | 324 | 345 | 1876 | 345 | 464 | 342 | 121 | 345 | 453 | 126 | 278 |
| Chr05 | 356 | 453 | 321 | 345 | 1475 | 245 | 241 | 231 | 242 | 257 | 165 | 189 |
| Chr06 | 364 | 456 | 135 | 464 | 245 | 1532 | 234 | 464 | 124 | 467 | 235 | 467 |
| Chr07 | 234 | 543 | 157 | 342 | 241 | 234 | 1653 | 356 | 345 | 135 | 463 | 234 |
| Chr08 | 457 | 247 | 145 | 121 | 231 | 464 | 356 | 1821 | 465 | 452 | 435 | 321 |
| Chr09 | 796 | 467 | 187 | 345 | 242 | 124 | 345 | 465 | 1209 | 421 | 345 | 342 |
| Chr10 | 421 | 564 | 243 | 453 | 257 | 467 | 135 | 452 | 421 | 1342 | 453 | 234 |
| Chr11 | 456 | 865 | 331 | 126 | 165 | 235 | 463 | 435 | 345 | 453 | 1768 | 432 |
| Chr12 | 321 | 345 | 254 | 278 | 189 | 467 | 234 | 321 | 342 | 234 | 432 | 1022 |
comparative example 1
Using the same immobilized cell starting material as in example 1, a library was constructed by the following procedure:
(1) after treatment with SDS, Triton, the supernatant was removed by brief centrifugation, the pellet resuspended in restriction enzyme Buffer, 1ul of Hind III-HF restriction enzyme was added and cleaved overnight at 37 ℃ on a rotary homogenizer. After the enzyme digestion is finished, the subsequent reaction is directly carried out without using the restriction enzyme inactivated at the high temperature of 65 ℃.
(2) The filling-in reaction is carried out at a low temperature of 23 ℃, and biotin-labeled nucleotides which are complementary with bases at the inner side of the tail end of the restriction enzyme cutting site of the restriction endonuclease are selected for labeling in the labeling process so as to improve the labeling effect.
(3) And centrifuging the filling product at the low temperature of 4 ℃ for 2min at 500g, removing supernatant, re-suspending the precipitate by using 1 XT 4 DNAILigaseBuffer, performing blunt end connection in a 250 mu L connection system according to the use amount of ligase of 1-2 Cohesiveunit/mu L, and connecting for 4-8 h at the temperature of 16 ℃.
(4) Adding NaCl with the final concentration of 200mmol/L and proteinase K with the final concentration of 1 mu g/mu L into the ligation product, performing decrosslinking at 65 ℃ overnight, treating with RNaseA to remove RNA, performing ethanol precipitation to recover DNA, performing secondary purification by using an QIAGENDNA recovery kit, and measuring the concentration of NanoDropND-1000. Electrophoresis is carried out on genomic DNA, restriction enzyme products, ligation products and the like respectively, and the detection and quality control of the enzyme digestion and ligation effects are carried out through the size of bands and the like.
(5) The purified and recovered Hi-C ligation product preferentially activates the exonuclease activity of T4DNA polymerase at low temperature of 12 ℃ and under the conditions of incomplete substrate nucleotides and the like, and biotin at the end of the unligated fragment is removed.
(6) And (3) carrying out ultrasonic crushing on the Hi-C sample, fragmenting the Hi-C sample into DNA with the size of 200-300 bp, slowly separating the sample through agarose gel electrophoresis, cutting gel for recovery, purifying the sample through QIAGENDNA recovery kit, and quantifying NanoDropND-1000.
(7) And (3) using the prewashed streptavidin C1beads to recover the DNA with the biotin label through the steps of uniform mixing, magnetic frame enrichment, supernatant removal, washing and re-enrichment and the like. In order to facilitate the conversion of the buffer system, the subsequent steps of end repair, adding "A" and connecting with Adapter are carried out on the magnetic beads with captured DNA.
(8) Taking 200ul Hi-C library template, using KAPAHiFi polymerase to perform PCR amplification for 10 fixed cycles, and obtaining the Hi-C library after electrophoresis, gel cutting and purification of amplification products.
(9) And selecting a DNA fragment with a biotin label, and sequencing by utilizing an Illumina process. The number of sites for the interaction found after analysis is shown in Table 2.
Table 2:
| Chr01 | Chr02 | Chr03 | Chr04 | Chr05 | Chr06 | Chr07 | Chr08 | Chr09 | Chr10 | Chr11 | Chr12 | |
| Chr01 | 645 | 76 | 87 | 60 | 87 | 45 | 65 | 91 | 53 | 63 | 80 | 31 |
| Chr02 | 76 | 367 | 53 | 57 | 55 | 50 | 53 | 62 | 91 | 63 | 93 | 50 |
| Chr03 | 87 | 53 | 574 | 46 | 50 | 45 | 68 | 63 | 70 | 64 | 69 | 42 |
| Chr04 | 60 | 57 | 46 | 532 | 46 | 69 | 42 | 68 | 71 | 83 | 57 | 25 |
| Chr05 | 87 | 55 | 50 | 46 | 478 | 54 | 35 | 76 | 48 | 65 | 79 | 47 |
| Chr06 | 45 | 50 | 45 | 69 | 54 | 490 | 67 | 54 | 47 | 73 | 65 | 73 |
| Chr07 | 65 | 53 | 68 | 42 | 35 | 67 | 584 | 64 | 24 | 53 | 75 | 45 |
| Chr08 | 91 | 62 | 63 | 68 | 76 | 54 | 64 | 357 | 75 | 35 | 36 | 78 |
| Chr09 | 53 | 91 | 70 | 71 | 48 | 47 | 24 | 75 | 689 | 76 | 56 | 59 |
| Chr10 | 63 | 63 | 64 | 83 | 65 | 73 | 53 | 35 | 76 | 590 | 46 | 54 |
| Chr11 | 80 | 93 | 69 | 57 | 79 | 65 | 75 | 36 | 56 | 46 | 685 | 67 |
| Chr12 | 31 | 50 | 42 | 25 | 47 | 73 | 45 | 78 | 59 | 54 | 67 | 579 |
from the number of the genomic interaction sites obtained from tables 1 and 2, the library construction method of the present application is not only simple in process, but also has great advantages in the number of the interaction sites that the constructed library can represent.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: this application constructs Hi-C libraries by ligating two adjacent cut ends of the cross-linked DNA together using a foreign known DNA sequence and performing a second cut with another restriction enzyme (e.g., MspI) (note that the primer annealing product cannot carry the cleavage site of the second restriction enzyme) to generate DNA fragments of appropriate size; and extracting and purifying after the second enzyme digestion, connecting the cut ends by using DNA ligase to form a circular chimeric molecule, and continuously extracting and purifying. The generated circular chimeric molecule is subjected to reverse PCR by designing a pair of reverse primers on the previously ligated primer annealing products, and the generated PCR product can be used as a Hi-C library. The whole genome interaction library constructed by the method can generate a large amount of paired terminal fragments for analysis by performing double-terminal sequencing on the whole genome interaction library constructed by the method by using a high-throughput sequencing method, and compared with the fragments generated by the library constructed by the conventional method, the method has the advantages of more abundant types of fragments and more complete interaction sites capable of being detected.
Moreover, in the Hi-C library constructed by the existing method, non-target results exist in the results obtained by high-throughput sequencing, and in the application, the target fragments are subjected to reverse amplification and then high-throughput sequencing, so that the existence of errors is greatly reduced, and the detected interaction sites are more accurate.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A method for constructing a genome-wide interaction library, the method comprising:
step S1, connecting the ends of the DNA-protein cross-linked compound generated by enzyme digestion by using a primer sequence to form a ring-shaped compound;
step S2, performing decrosslinking on the circular compound to obtain circular DNA; and
step S3, carrying out fragmentation library construction on the circular DNA to obtain the whole genome interaction library;
the primer sequence is a sequence with the two ends provided with enzyme cutting sticky ends, the enzyme cutting sticky ends are the same as the sticky ends generated by the enzyme cutting of the first endonuclease, and the primer sequence does not contain the enzyme cutting sites of the second endonuclease.
2. The building method according to claim 1, wherein the step S1 includes:
step S11, performing first enzyme digestion treatment on the DNA-protein cross-linked compound by using a first endonuclease to obtain a first enzyme digestion product;
and step S12, annealing and connecting the first enzyme digestion product and the primer sequence to obtain the circular compound.
3. The building method according to claim 2, wherein the step S3 includes:
step S31, performing secondary enzyme digestion treatment on the circular DNA by adopting second endonuclease treatment to obtain a second enzyme digestion product;
step S32, carrying out cyclization treatment on the second enzyme digestion product to obtain a cyclization product;
and step S33, performing reverse PCR on the cyclization product by using the primer sequence to obtain the whole genome interaction library.
4. The method of any one of claims 1 to 3, wherein the primer sequence is 10 to 40bp in length.
5. The building method according to claim 3, wherein the step S33 includes:
designing two amplification primers in opposite directions on the primer sequence;
and carrying out reverse PCR on the cyclization product by using the amplification primer to obtain the whole genome interaction library.
6. The method of constructing according to claim 1 or 2, wherein before performing the step S1, the method of constructing further comprises a step of obtaining a DNA-protein cross-linked complex, the step of obtaining a DNA-protein cross-linked complex comprising:
fixing a cell sample to be detected by using formaldehyde to obtain fixed cells;
and (3) cracking the fixed cells to obtain the DNA-protein cross-linked complex.
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