CN1159456C - Method for screening DNA fragments for genetic variation and protein binding - Google Patents

Abstract

The present invention mainly includes methods for preparing DNA fragments, detecting DNA fragments combined with proteins and identifying gene variation. The method of detecting protein-bound DNA fragments: Genomic DNA is digested with endonuclease or PCR amplification with primers to prepare fragments, and the ends of these DNA fragments are modified with groups to make covalent interaction with the corresponding solid phase carrier surface. Linking, using a microarray spotting device to spot various DNA fragments on the surface of a solid phase carrier and covalently cross-linking them to prepare a DNA chip, using this DNA chip to screen protein-bound DNA fragments.

Description

The method of the dna fragmentation of screening-gene variation and protein bound

What the present invention relates to is the method for the dna fragmentation of a kind of identified gene variation and screening protein bound.Belong to field of molecular biotechnology.

Protein is the key of gene expression regulation, chromosome structure structure, dna replication dna and reparation, genetic transcription etc. with combining of DNA.Set up the method for some identification of dna and protein bound.In vitro method has gel retardation assasy, molecular sieve filtration experiment etc.; Intracorporal method has the monobasic hybrid method, exactly a reporter gene is placed the downstream of one section dna fragmentation that may combine with protein, in same cell with possible DNA conjugated protein with the proteic activation domain amalgamation and expression of genetic transcription, if this protein combines with this segment DNA, reporter gene will be expressed.The disadvantage of these methods is that workload is big, and it is conjugated protein once can only to analyze a kind of DNA; Another shortcoming is that blindness is big.Biology field is badly in need of a kind of method that once can screen the conjugated protein institute of DNA bonded dna fragmentations all in the organism.Polymorphism Analysis is an important research field of mankind nowadays genetics research, and the technology of carrying out this analysis has determined dna sequence, normal people's gene also to carry out gradient variable electrophoresis (SSCP), restricted enzyme cutting spectrum analysis etc. after the pairing of patient's allelotrope with heredity.The workload of these technology is big; And when the genetic background lack of knowledge, carry out linkage analysis, otherwise blindness is big; For disease of multifactorial inheritance, be difficult to carry out Analysis and Identification with these methods.Method with gene chip analyzing gene polymorphism has appearred in the document recently, the rope E.M.1996.DNA chip that pauses: extensive oligonucleotide hybridization analytical sequence.The genetics progress.12(3)：110-115。This method is exactly that the known dna fragmentation of sequence is fixed in the surface of solid phase carrier with the form of oligonucleotide, and the allelotrope of individuality to be measured fluorophor mark is then with the DNA chip hybridization.This technology aspect the polymorphism that detects the sequence known of great use, but polymorphism that can not the analytical sequence unknown gene, and need synthetic a large amount of oligonucleotide fragment, expense is big, the semaphore that this method produces is big, the difficulty height of analysis.

The objective of the invention is to be to overcome the deficiencies in the prior art, the method for the dna fragmentation of a kind of screening-gene variation and protein bound is provided.

Technical scheme of the present invention is as follows: mainly comprise the method for dna fragmentation of the method, detection and the protein bound that prepare dna fragmentation and the method for identified gene variation.

1. the method that detects the protein bound dna fragmentation is as follows: genomic dna is prepared the different dna fragmentation that comprises whole genome with digestion with restriction enzyme or with the pcr amplification that specific primer carries out, end with these dna fragmentations of specificity base group modification, enable to carry out covalent cross-linking with corresponding surface of solid phase carriers, with microarray point sample equipment with various dna fragmentation point samples in surface of solid phase carriers and covalent cross-linking it, be prepared into the DNA chip, the method of screening the dna fragmentation of protein bound with this DNA chip is that (a) directly is incubated the DNA chip with fluorescently-labeled protein, and protein institute bonded dna fragmentation is represented in the fluorescent signal site of chip; Or (b) the direct and protein insulation with dna fragmentation, to cut with the DNase enzyme then, the dna fragmentation that combines with protein can not cut by the DNase enzyme, with isotropic substance or the protected dna fragmentation of fluorophor mark, last and DNA chip hybridization; Perhaps directly and the DNA chip hybridization with protected dna fragmentation; with the dna fragmentation of dna ligase or RNA ligase enzyme or archaeal dna polymerase enzymatic reaction mark and chip hybridization, isotropic substance signal on the chip or fluorescent signal are represented the dna fragmentation with protein bound again.

2. the method for identified gene variation is as follows: the DNA chip production method is the same, but follow-up method is slightly different.The dna fragmentation (a kind of be normal type or be called standard type that another kind is called anomaly or type to be measured) that derives from Different Individual is hybridized, and the hybridization product has three kinds: (1) two chain all derives from normal type, and DNA matches fully; Article (2) two, chain all derives from anomaly, and DNA matches fully; Article (3) one, chain derives from normal type, another chain derives from anomaly, the DNA base of variation place mismatches maybe and can not match, therefore can combine with dna repair protein such as MUT S, after will hybridizing product and MUT S combining, cut with the DNase enzyme, base mismatches or the dna fragmentation that is unworthy of does not have digested, with isotropic substance or the protected dna fragmentation of fluorescent mark group mark, again with the DNA chip hybridization; Perhaps directly with protected dna fragmentation and DNA chip hybridization; use RNA ligase enzyme or dna ligase or archaeal dna polymerase enzymatic reaction label isotope or fluorophor then; isotropic substance on the DNA chip or fluorescent signal are represented the dna fragmentation at genovariation place; another kind method is that the dna fragmentation of anomaly and DNA chip are hybridized; hybridization back and isotropic substance or fluorescently-labeled MUT S insulation; perhaps with after being incubated with the crosslinked MUT S of colour developing enzyme develop the color, the dna fragmentation at genovariation place is represented in the model site on the chip.

Significance effect of the present invention and substantive distinguishing features show: (1) can screen the dna fragmentation that combines with protein on a large scale, the dna fragmentation at identified gene variation place; (2) processing ease, data are comprehensive.(3) signal is simple, is easy to analyze.Therefore can promote the research and development of being correlated with greatly.

Below in conjunction with accompanying drawing the embodiment of the invention is further described:

Fig. 1 normal type genomic dna carries out the synoptic diagram of pcr amplification.

Fig. 2 normal type genomic dna restriction enzyme digestion prepares the synoptic diagram of dna fragmentation.

The synoptic diagram of the laggard performing PCR amplification of Fig. 3 normal type genomic dna restriction enzyme digestion.

The synoptic diagram of Fig. 4 DNA chip preparation.

Fig. 5 DNA chip directly and the synoptic diagram of protein bound.

Behind the dna fragmentation mark of Fig. 6 and protein bound with the synoptic diagram of DNA chip hybridization.

The synoptic diagram of mark again behind the dna fragmentation of Fig. 7 and protein bound and the DNA chip hybridization.

Behind Fig. 8 DNA to be measured and the DNA chip hybridization with MUT S bonded synoptic diagram.

Fig. 9 mismatches behind the dna fragmentation mark synoptic diagram with the DNA chip hybridization.

Figure 10 mismatches the synoptic diagram of dna fragmentation and the laggard row labels of DNA chip hybridization.

1. can prepare dna fragmentation and make the DNA chip with following method:

As shown in Figure 1, → forward primer, ← reverse primer.The standard type genomic dna directly carries out pcr amplification with a cover Auele Specific Primer, prepares the dna fragmentation that thousands of kinds are suitable for the chip preparation.

As shown in Figure 2, normal type genomic dna `1 step use digestion with restriction enzyme, the 2nd step usefulness gel technique technology or other technical point from, be prepared into various dna fragmentations.Use specificity base group modification end then, make it to be suitable for behind the point sample covalent cross-linking with the solid phase surface group.Through suitable approach such as technical points such as electrophoresis, chromatography from, prepare diversified dna fragmentation.Modify the end of these dna fragmentations with suitable chemical group, make it to be suitable for being used for preparing the DNA chip.

As shown in Figure 3, the normal type genomic dna `1 step is used digestion with restriction enzyme, the 2nd step enzyme is cut product and is connected with suitable LINKER, the 3rd step is with matching with corresponding LINKER, but 3 ' end has the primer of extra 4 Nucleotide carries out pcr amplification, therefore has 256 * 256 kinds of combination of primers to carry out the PCR reaction.

As shown in Figure 4, with the dna fragmentation that method obtained shown in Fig. 1-3, by hand or microarray point sample equipment point sample in suitable substrate surface and carry out covalent cross-linking reaction, preparation DNA chip.

2. can be with the dna fragmentation of following method screening with protein bound:

As shown in Figure 5, with radio isotope or fluorophor mark or with the crosslinked conjugated protein insulation of DNA of colour developing enzyme, after DNA and the protein bound, flush away boiling delegation bonded protein, detect the combination of proteins site with test set, if DNA is conjugated protein is that enzyme is crosslinked, develops the color earlier and afterwards detects.Signal appears in the corresponding positions on the chip ●, sort signal is represented protein institute bonded dna fragmentation.

As shown in Figure 6, the 1st step protein combines in liquid phase with DNA, and the 2nd step cut with the DNase enzyme; the 3rd step was reclaimed the dna segment of protein bound; the 4th step was protected with radio isotope or fluorophor with radio isotope or fluorescent mark, shielded dna fragmentation, the 5th step and DNA chip hybridization.Chip signal ● the dna fragmentation of expression and protein bound.

As shown in Figure 7; the 1st step protein combines in liquid phase with DNA; the 2nd step cut with the DNase enzyme then; the 3rd step was reclaimed protected dna segment; the 4th step was carried out nucleic acid hybridization with the DNA chip; carry out isotropic substance or fluorescent mark with enzymatic reaction then, shielded dna fragmentation directly and the DNA chip hybridization, then in the enzymatic reaction of RNA ligase enzyme or dna ligase or archaeal dna polymerase with the hybridization site on radio isotope or the fluorophor tagging chip.Chip signal ● the dna fragmentation of expression and protein bound.

3. can make a variation with following method identified gene:

As shown in Figure 8, dna fragmentation to be measured is at first hybridized with the DNA chip, (it is with radio isotope or fluorophor mark that the DNA DNA plerosis is repaired albumen with dna repair protein MUT S then, perhaps crosslinked with suitable colour developing enzyme) combine (note: with must show reaction after the crosslinked dna repair protein of colour developing enzyme and the DNA chips incorporate), chip signal ● the dna fragmentation at the place of representing to make a variation.

As shown in Figure 9; the 1st step normal type DNA and anomaly DNA are hybridized in liquid phase; two chains of double-stranded 1 derive from normal type, double-stranded 2 a normal type, and another is an anomaly; two chains of double-stranded 3 all derive from anomaly; the 2nd step combined with dna repair protein such as MUT S again, and the 3rd step cut with the DNase enzyme, and the 4th step was reclaimed the dna segment of protection; with radio isotope or the protected dna fragmentation of fluorophor mark, the 5th step and DNA chip hybridization.Chip signal ● the dna fragmentation at expression variation place.

As shown in figure 10; the 1st step normal type DNA and anomaly DNA hybridization earlier; the 2nd step combined with dna repair protein such as MUT S again; the 3rd step cut with the DNase enzyme; direct and the DNA chip hybridization of shielded dna fragmentation of the 4th step, last dna fragmentation of in RNA ligase enzyme or dna ligase or archaeal dna polymerase enzymatic reaction, hybridizing with radio isotope or fluorophor mark.Chip signal ● the dna fragmentation at expression variation place.

4, the detection of DNA chip signal:

The signal that labelled with radioisotope produces can use X-mating plate autography or isotropic substance imager or scanner to detect, and the signal that fluorophor mark or other luminophore produce can detect with optical detection apparatus.

Claims (1)

1. A method for screening gene variation and protein-bound DNA fragments, mainly comprising a method for preparing DNA fragments, a method for detecting protein-bound DNA fragments and a method for identifying gene variation, characterized in that the method for detecting protein-bound DNA fragments As follows: Genomic DNA is digested with restriction endonuclease or PCR amplification with specific primers to prepare DNA fragments containing the entire genome, and the ends of these DNA fragments are modified with specific groups so that they can be compatible with the corresponding The surface of the solid phase carrier is covalently cross-linked, and various DNA fragments are spotted on the surface of the solid phase carrier with a microarray spotting device and covalently cross-linked to prepare a DNA chip, which is used to screen protein-bound DNA The fragmentation method is (a) incubating the DNA chip directly with the fluorescently labeled protein, and the fluorescent signal site of the chip indicates the DNA fragment bound to the protein; or (b) directly incubating the DNA fragment with the protein, and then digesting it with DNase, Label the protected DNA fragments with isotopes or fluorescent groups, and finally hybridize with DNA chips; or directly hybridize the protected DNA fragments with DNA chips, and then use DNA ligase or RNA ligase or DNA polymerase enzymatic reaction to label and DNA fragments hybridized to the chip, isotopic signals or fluorescent signals on the chip indicate DNA fragments bound to proteins.

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