CN1435491A - Polymerase chain reaction mediated DNA bindin detection reagent and use method - Google Patents
Polymerase chain reaction mediated DNA bindin detection reagent and use method Download PDFInfo
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- CN1435491A CN1435491A CN 03112916 CN03112916A CN1435491A CN 1435491 A CN1435491 A CN 1435491A CN 03112916 CN03112916 CN 03112916 CN 03112916 A CN03112916 A CN 03112916A CN 1435491 A CN1435491 A CN 1435491A
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Abstract
A reagent for detecting the PCR mediated DNA bindin is a group of dual-chain oligonucleotide sequences. The middle part of each sequence contains the recognizing sequences of the DNA for the DNA bindin to be detected and the particular restriction endonuclease. Its application method includes adding the reagent to the solution of bindin to be detected for incubation, adding relative restriction endonuclease for sufficient digestion, adding proteinase K to remove protein, PCR amplifying, and detecting the resultant to obtain the information about said DNA bindin.
Description
Technical field
What the present invention relates to is a kind of conjugated protein detection reagent of DNA and using method of polymerase chain reaction mediation.The DNA here is conjugated protein to be meant that mainly to have sequence-specific DNA conjugated protein, as regulatory factor of relating in the genetic expression etc.
Technical background
DNA and proteinic sequence-specific identification and interacting plays an important role in the biological gene expression regulation, the generation of nearly all disease and development all with cell in DNA protein-bonded relevant unusually.At present, the protein-bonded research of DNA more and more is subjected to people's attention, becomes one of important content of genome and proteome research.Therefore, the detection of dna binding protein dna in pair cell or the diseased tissue, particularly high-throughout detection is vital.
Be used for researching DNA at present and method of protein is a lot, as gel shift rate analysis (EMSA), footprinting (foot-printing) etc.Though these conventional methods have been brought into play great function in the protein-bonded research of sequence-specific DNA, their workloads greatly, very time-consuming, cause the pollution of radioactive substance in the operation easily, but also can't carry out high-throughout detection.It is conjugated protein that the method for having reported a kind of novel molecular beacon (Molecular Beacon) recently on " Nature Biotechnol " (Nature Biotechnology) magazine detects DNA, though this method has been avoided the pollution of radioactive substance, but still can't carry out high throughput testing.Gene chip, claim the DNA chip again, to carry out advantages such as large-scale operation application has widely been arranged in methods such as expression of gene spectrum analysis, single base polymorphisms (SNP) detections at expression of gene spectrum analysis, single base polymorphisms because it convenient, fast can carry out advantage such as large-scale operation.U.S. Affymetrix company and the Wu Jian of Southeast China University have successively developed some novel double-stranded DNA chips in male laboratory, can be to the conjugated protein large-scale detection of carrying out of a plurality of DNA.But these methods must be carried out the fluorescence molecule mark to protein before detection, may cause losing of low abundance proteins like this in the process of mark, are unfavorable for improving detection efficiency and sensitivity.
Used polymerase chain reaction (PCR) technology of the present invention mainly is as a kind of selectivity amplification in vitro method.
Summary of the invention
The objective of the invention is to provide a kind of conjugated protein detection reagent of DNA and using method of polymerase chain reaction mediation at above-mentioned weak point, abundance in order to a certain in the detection cell or tissue or multiple dna binding protein dna also can be used for detecting the variation of the conjugated protein content of DNA in normal and the diseased tissue.Its principle is: (double chain oligonucleotide designs the protein-bonded recognition site of restriction endonuclease such as EcoRI and DNA on dsDNA) simultaneously, and two sites are closely linked to each other at a double-stranded DNA.Earlier double chain oligonucleotide is hatched certain hour with cell extract to be detected, then this DNA-protein complex is cut with the abundant enzyme of EcoRI restriction endonuclease.Because sterically hindered effect, combining proteinic oligonucleotide sequence can't be digested.Not digested oligonucleotide sequence is separated from mixture, carry out pcr amplification with the single primer that has marker, or in amplification, mix the free base of mark fluorescent group; To contain with amplified production complementary probe points and make chip on slide, amplified production and chip hybridization carry out hybridization signal and detect or DIRECT GEL electrophoresis detection PCR product.The protein-bonded content of DNA detected in the power of signal or electrophoretic band and the protein mixture is relevant.
The conjugated protein detection reagent of DNA and the using method of a kind of polymerase chain reaction mediation of the present invention take following scheme to realize:
A kind of conjugated protein detection reagent of DNA of polymerase chain reaction mediation, it is characterized in that at one group of detected dna binding protein dna, design and prepare one group of double chain oligonucleotide sequence, wherein the middle portion of each double chain oligonucleotide sequence comprises the recognition sequence of a kind of protein-bonded specific recognition sequence of DNA to be detected and specific DNA restriction enzyme, and the two is closely linked to each other; Sequence in double chain oligonucleotide except that the DNA binding protein recognition sequence is consistent.
A kind of using method of the conjugated protein detection reagent of DNA of polymerase chain reaction mediation, it is characterized in that (1) adds above-mentioned double chain oligonucleotide detection reagent in the solution that contains dna binding protein dna hatches, and makes the abundant combination of the conjugated protein double chain oligonucleotide corresponding with it of DNA in the solution; (2) adding fully digests with the corresponding restriction enzyme of restriction enzyme site in above-mentioned solution, and the double chain oligonucleotide of unbinding protein is cut off by restriction endonuclease; (3) cut the protein that adds in the product in the Proteinase K complete digestion product at enzyme; (4) digestion product carries out pcr amplification with primer; (5) by detecting above-mentioned PCR product, obtain the protein-bonded information of a plurality of DNA in the solution.
DNA restriction enzyme in a kind of using method of the protein-bonded detection reagent of DNA of PCR mediation comprises flat terminal restriction endonuclease (as: RsaI) and sticky end restriction endonuclease (as: EcoRI).
A pair of or PCR primer with the terminal complementary of double chain oligonucleotide sequence in the using method of the conjugated protein detection reagent of DNA of a kind of polymerase chain reaction mediation is public PCR primer, promptly can the while increase one group of double chain oligonucleotide out with a pair of or a public primer.
PCR product in a kind of using method of the conjugated protein detection reagent of DNA of polymerase chain reaction mediation detects and can adopt single primer to increase, or amplified production is directly carried out detected through gel electrophoresis with the method for quantitative PCR; Also can adopt the micro-array chip technology to carry out PCR product detection (1) and be used to detect the protein-bonded double chain oligonucleotide PCR of DNA product at each bar, design and preparation and its part complementary single strand oligonucleotide probes, and be fixed on the solid substrate; (2) in amplification procedure, introduce marker; (3) amplified production is hybridized the amplified production sequence that wash-out is not hybridized with the chip for preparing; (4) hybridization signal to chip detects.
In the using method of the conjugated protein detection reagent of DNA of a kind of polymerase chain reaction mediation enzyme being cut the marker that product carries out introducing in the pcr amplification process is fluorescence molecule group, vitamin H, nano particle etc.
The oligonucleotide probe institute fixed solid substrate of preparation chip can be microballoon, nylon membrane, cellulose acetate membrane, plastics, rubber, pottery or sheet glass etc. in a kind of using method of the protein-bonded detection reagent of DNA of polymerase chain reaction mediation.
PCR product based on micro-array chip in a kind of using method of the protein-bonded detection reagent of DNA of polymerase chain reaction mediation detects the detection method that can adopt quantification, conjugated protein with a certain DNA of concentration known earlier, set up the typical curve of protein concentration and strength of signal according to the method described above, again the concentration of this protein in sample is carried out detection by quantitative.
In the using method of the conjugated protein detection reagent of DNA of a kind of polymerase chain reaction mediation based on the PCR product of micro-array chip detect also can adopt diversity ratio method detect PCR product (1) get two kinds of different states as normal and morbid state, drug treating tissue or cells front and back or the different periods of growing, extract cell extract, the cell extract of two kinds of different states is mixed with the double chain oligonucleotide detection reagent of design respectively hatch; (2) under identical conditions, carry out pcr amplification, mix respectively in the amplification different fluorescent emission (or exciting) wavelength of mark as the Brdurd Triphosaden (dUTP) of fluorescence molecule Cy3 and Cy5 mark etc.; (3) two kinds of amplified production equal-volumes mix back and chip hybridization; (4) detect the difference of the protein-bonded content of certain DNA in sample according to the Two Colour Fluorescence method.
Oligonucleotide microarray chip in a kind of using method of the conjugated protein detection reagent of DNA of polymerase chain reaction mediation can be used for the protein-bonded detection of a plurality of DNA.
A kind of using method of the conjugated protein detection reagent of DNA of polymerase chain reaction mediation also can be carried out the protein-bonded detection of DNA of (in vivo) in the cell, its process is for to transfer to the double chain oligonucleotide fragment in the cell by the method for microinjection or electrotransfer, cultivate cryogenic pulverization cell after for some time, make that DNA in the thin bag is conjugated protein to carry out combining of composition with double chain oligonucleotide.
The present invention has developed a kind of conjugated protein detection reagent of DNA and using method thereof of polymerase chain reaction mediation, in order to detect the abundance of dna binding protein dna in the cell or tissue, especially can be used for detecting the variation of the conjugated protein content of DNA in normal and the diseased tissue.One of advantage of this method is not need protein is carried out fluorescent mark, and proteinic mole number is converted into the mole number of oligonucleotide fragment, by the primer PCR amplification of mark fluorescent, carries out signal and amplifies then, detects with chip hybridization again; Simultaneously also have high throughput testing, radiocontamination with low cost, simple and efficient, no and can carry out advantage such as the interior protein-bonded abundance detection of DNA of living organisms, be highly suitable for the prediction of disease, the advantages such as proteic abundance detection of pathogenesis, be highly suitable for research fields such as the discussion of prediction, pathogenesis of disease and drug screening.
Description of drawings
The invention will be further described below with reference to accompanying drawing.
Fig. 1 is the design diagram of double chain oligonucleotide of the present invention.
Fig. 2 is the public primer design synoptic diagram of the present invention.
Fig. 3 is using method synoptic diagram of the present invention (comprising Fig. 3-1,3-2,3-3,3-4).
Fig. 4 is the synoptic diagram that detects the PCR product in the using method of the present invention with gel electrophoresis.
Fig. 5 is the different concns in the using method of the present invention and the typical curve synoptic diagram of strength of signal.
Embodiment
With reference to accompanying drawing 1, a kind of conjugated protein detection reagent of DNA of polymerase chain reaction mediation, it is characterized in that at one group of detected dna binding protein dna, design and prepare one group of double chain oligonucleotide sequence, wherein the middle portion of each double chain oligonucleotide sequence comprises a kind of protein-bonded specific binding site of DNA to be detected and the recognition sequence of EcoRI, and the two is closely linked to each other; Sequence in double chain oligonucleotide except that the DNA binding protein recognition sequence is consistent.Embodiment 1: the preparation of single primer extension method 1 double chain oligonucleotide
According to the protein-bonded specific recognition sequences Design of each DNA and preparation two complementary (40～100 base) oligonucleotide (as Fig. 1), the recognition sequence of combination of proteins sequence with restriction endonuclease such as EcoRI closely linked to each other.The contained restriction enzyme site to 3 of each bar double chain oligonucleotide here ' terminal sequence is consistent.2, the preparation of single primer
The restriction enzyme site to 3 that contains according to the double chain oligonucleotide that designs ' terminal concensus sequence, single primer (as Fig. 2) of 20 bases of design, and with its 5 ' end introducing marker (as fluorescence molecule group, vitamin H, nano particle etc.).This primer is a universal primer, and the protein-bonded double chain oligonucleotide of DNA that contains of all designs all is suitable for.3, the preparation of chip (as Fig. 3-4)
According to 25nt of the specificity of the protein-bonded recognition sequence of each DNA design and can with single primer extension product complementary single strand oligonucleotide probes, one-NH of its 5 ' end mark
2, these probes are fixed on the slide of aminosilaneization the preparation chip by the point sample method according to certain array.4, the acquisition of test material
The object of Jian Ceing is chosen different tissues or cultured cells as required.Must carry out quantitatively tissue or cell before the preparation cell extract, under peer-level, detect guaranteeing.After the tissue of equivalent or cell cleaned with phosphate buffered saline buffer, low-temperature homogenate, centrifugal muscle and the cytoskeletons etc. of removing are to obtain cell extract.5, the protein-bonded testing process of DNA (as Fig. 3)
(1) reaction of the protein bound in double chain oligonucleotide and the cell extract (as Fig. 3-1)
A certain amount of cell extract is mixed with quantitative double chain oligonucleotide, and 25 ℃ in conjunction with 30 minutes.The all corresponding double chain oligonucleotide of each protein to be measured.
(2) endonuclease reaction of DNA-protein complex (as Fig. 3-2)
Get quantitative above-mentioned reactant,, and be combined with proteinic double chain oligonucleotide owing to be subjected to sterically hindered effect and fail digested with 37 ℃ of the EcoRI enzymes complete digestion free double chain oligonucleotide that spends the night.Add an amount of Proteinase K then, 55 ℃ are spent the night, and protein is removed in centrifugal digestion, reclaim the two strands that enzyme is not cut.(3) single primer PCR amplifying doulbe-chain oligonucleotide (as Fig. 3-3)
With the above-mentioned not digested digestion of the single primer PCR amplification that has marker and with the double chain oligonucleotide sequence of protein separation.(4) hybridization of chip (as Fig. 3-4)
With above-mentioned amplified production and the good chip hybridization of prepared beforehand.(5) detection of hybridization signal and data analysis
Detect with corresponding means according to different markers.As fluorescent microscope, Laser Scanning Confocal Microscope, chip scanner etc.Scanning result compares with the equal graphic representation of mark of strength of signal-protein concn of doing according to such scheme with the protein of concentration known in advance, draws detected result (as Fig. 4).
Embodiment 2: the Two Colour Fluorescence method
The preparation of the preparation of double chain oligonucleotide, the preparation of single primer, chip and the association reaction of protein and double chain oligonucleotide and EcoRI endonuclease reaction are with embodiment 1.Test material is two kinds of different states (normal and morbid state, the drug treating front and back or the different periods of growing) tissue or cells, extract cell extract, the cell extract of two kinds of different states is mixed with the double chain oligonucleotide detection reagent of design respectively hatch; In amplification, mix the Brdurd Triphosaden (dUTP) of fluorescence molecule Cy3 and Cy5 mark respectively; Two kinds of amplified production equal-volumes mix back and chip hybridization; Detect the difference of the protein-bonded content of certain DNA in sample according to the Two Colour Fluorescence method.Embodiment 3:PCR amplified production electrophoretic method
Pcr amplification and former step thereof are with embodiment 1 or 2.With a certain amount of and carry out gel electrophoresis simultaneously with corresponding to oligonucleotide sequence of amplified production and pcr amplification product.Judge the variation of protein-bonded amount of DNA or abundance according to the power of electrophoretic band.(as Fig. 5)
Claims (10)
1, a kind of protein-bonded detection reagent of DNA of polymerase chain reaction mediation, it is characterized in that at one group of detected dna binding protein dna, design and prepare one group of double chain oligonucleotide sequence, wherein the middle portion of each double chain oligonucleotide sequence comprises the recognition sequence of a kind of protein-bonded specific recognition sequence of DNA to be detected and specific DNA restriction enzyme, and the two is closely linked to each other; Sequence in double chain oligonucleotide except that the DNA binding protein recognition sequence is consistent.
2, the using method of the protein-bonded detection reagent of DNA of the described a kind of polymerase chain reaction mediation of claim 1, it is characterized in that (1) adds above-mentioned double chain oligonucleotide detection reagent in the solution that contains dna binding protein dna hatches, and makes the abundant combination of the conjugated protein double chain oligonucleotide corresponding with it of DNA in the solution; (2) adding fully digests with the corresponding restriction enzyme of restriction enzyme site in above-mentioned solution, and the double chain oligonucleotide of unbinding protein is cut off by restriction endonuclease; (3) cut the protein that adds in the product in the Proteinase K complete digestion product at enzyme; (4) digestion product carries out PCR amplification with primer; (5) by detecting above-mentioned polymerase chain reaction product, obtain the protein-bonded information of a plurality of DNA in the solution.
3, according to the using method of the protein-bonded detection reagent of DNA of the described a kind of polymerase chain reactions mediation of right 2, it is characterized in that the DNA restriction enzyme comprises flat terminal restriction endonuclease and sticky end restriction endonuclease.
4, according to the using method of the protein-bonded detection reagent of DNA of the described a kind of polymerase chain reactions mediation of right 2, it is characterized in that a pair of or chain reaction primer of polymerase is public chain reaction primer of polymerase with the terminal complementary of double chain oligonucleotide sequence, promptly can the while increase one group of double chain oligonucleotide out with a pair of or a public primer.
5, according to the using method of the protein-bonded detection reagent of DNA of the described a kind of polymerase chain reactions mediation of right 2, it is characterized in that detecting the method for the product of polymerase chain reaction, adopt single primer to increase with quantitative PCR; Or amplified production carried out detected through gel electrophoresis; Or the product that adopts the micro-array chip technology to carry out polymerase chain reaction detects (1) is used to detect the protein-bonded double chain oligonucleotide polymerase chain reaction of DNA at each bar product, design and preparation and its part complementary single strand oligonucleotide probes, and be fixed on the solid substrate; (2) in the polymerase chain reaction expansion process, introduce marker;
(3) amplified production is hybridized the amplified production sequence that wash-out is not hybridized with the chip for preparing; (4) hybridization signal to chip detects.
6,, it is characterized in that it is fluorescence molecule group, vitamin H, nano particle that enzyme is cut the marker that product carries out introducing in the PCR amplification process according to the using method of the protein-bonded detection reagent of DNA of the described a kind of polymerase chain reactions mediation of right 5.
7, according to the using method of the protein-bonded detection reagent of DNA of the described a kind of polymerase chain reactions mediation of right 5, the oligonucleotide probe institute fixed solid substrate that it is characterized in that preparing chip is microballoon, nylon membrane, cellulose acetate membrane, plastics, rubber, pottery or sheet glass.
8, according to the using method of the protein-bonded detection reagent of DNA of the described a kind of polymerase chain reactions mediation of right 5, it is characterized in that adopting the detection method of quantification based on the polymerase chain reaction product detection method of micro-array chip, conjugated protein with a certain DNA of concentration known earlier, set up the typical curve of protein concentration and strength of signal according to the method described above, again the concentration of this protein in sample is carried out detection by quantitative; Or adopt diversity ratio method to detect polymerase chain reaction product, in the difference comparison and detection method (1) get two kinds of different states as tissue or cells normal and morbid state, before and after the drug treating or the different periods of growing, extract cell extract, the cell extract of two kinds of different states is mixed with the double chain oligonucleotide detection reagent of preparation respectively hatch;
(2) under identical conditions, carry out PCR amplification, mix the deoxy-guanine Triphosaden of different fluorescent emission of mark or excitation wavelength in the amplification respectively; (3) two kinds of amplified production equal-volumes mix back and chip hybridization; (4) detect the difference of the protein-bonded content of certain DNA in sample according to the Two Colour Fluorescence method.
9, the using method of the conjugated protein detection reagent of DNA of a kind of polymerase chain reaction mediation according to claim 5 is characterized in that the oligonucleotide microarray chip is used for the protein-bonded detection of a plurality of DNA.
10, the using method of the protein-bonded detection reagent of DNA of right 1 described a kind of polymerase chain reaction mediation, it is characterized in that this method can carry out the protein-bonded detection of viable cell DNA, its process is for to transfer to the double chain oligonucleotide fragment in the cell by the method for microinjection or electrotransfer, cultivate cryogenic pulverization cell after for some time, make that intracellular DNA is conjugated protein to carry out combining of composition with double chain oligonucleotide.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104846083A (en) * | 2015-04-30 | 2015-08-19 | 华南农业大学 | Method for identifying combining interaction of protein and DNA (Deoxyribose Nucleic Acid) through RFLP (Restriction Fragment Length Polymorphism) |
| WO2021026693A1 (en) * | 2019-08-09 | 2021-02-18 | 宁芯(南京)生物医学技术研究院有限公司 | Characteristic nucleotide, probe, kit and method for identifying fermented cordyceps powder |
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2003
- 2003-03-05 CN CN 03112916 patent/CN1435491A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104846083A (en) * | 2015-04-30 | 2015-08-19 | 华南农业大学 | Method for identifying combining interaction of protein and DNA (Deoxyribose Nucleic Acid) through RFLP (Restriction Fragment Length Polymorphism) |
| WO2021026693A1 (en) * | 2019-08-09 | 2021-02-18 | 宁芯(南京)生物医学技术研究院有限公司 | Characteristic nucleotide, probe, kit and method for identifying fermented cordyceps powder |
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