CN1435492A - Chip for non-label detecting DNA bindin, preparation and use method thereof - Google Patents
Chip for non-label detecting DNA bindin, preparation and use method thereof Download PDFInfo
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- CN1435492A CN1435492A CN 03112917 CN03112917A CN1435492A CN 1435492 A CN1435492 A CN 1435492A CN 03112917 CN03112917 CN 03112917 CN 03112917 A CN03112917 A CN 03112917A CN 1435492 A CN1435492 A CN 1435492A
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Abstract
A chip for the non-label detection of DNA bindin has the dual-chain oligonucleotide probes designed according to the specific DNA recognizing sequence for the DNA bindin to be detected. The probe A is fixed to the substrate, and a label is introduced to the probe B. The probe B and the solution of the DNA bindin are inculated together and then hybridized with chip. If DNA bindin exists, the probes A and B have very high binding efficiency and the label of probe B can be detected at the position of probe A.
Description
Technical field
What the present invention relates to is that a kind of non-marked detects the protein-bonded chip of DNA and preparation thereof, application method.The conjugated protein regulatory factor that mainly is meant of the DNA here with special dna binding sequence row.This chip can detect simultaneously to a plurality of DNA are conjugated protein, have high-throughput, highly sensitive, directly perceived, preparation is simple, cost is low, need not advantage such as labelled protein.
Background technology
DNA and the identification of proteinic sequence-specific and interaction play an important role in the biological gene expression regulation, and the generation of nearly all disease is all protein-bonded relevant unusually with DNA with development.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.The detection of the amount of a certain or multiple proteins is vital in pair cell or the diseased tissue.
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, this method has been avoided the pollution of radioactive substance, easy to detect, but can't carry out the high-throughput operation.Gene chip claims the DNA chip again, because it convenient, fast can carry out advantage such as large-scale operation application has widely been arranged in methods such as expression of gene spectrum analysis, single base polymorphisms (SNP) detections.But the double-stranded DNA combination of conjugated protein of most of DNA and distinguished sequence.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.
Summary of the invention
The objective of the invention is provides a kind of non-marked to detect the protein-bonded chip of DNA and preparation thereof, application method at above-mentioned weak point, it has developed the abundance that a kind of cold biochip technology detects 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, also have simultaneously advantages such as high throughput testing, with low cost, simple and efficient, no radiocontamination, be highly suitable for research fields such as the discussion of prediction, pathogenesis of disease and drug screening.
A kind of non-marked detection protein-bonded chip of DNA of the present invention and preparation thereof, application method take following scheme to realize:
Non-marked of the present invention detects the protein-bonded chip of DNA, it is characterized in that structure has the double chain oligonucleotide probe, the sequence of double chain oligonucleotide probe designs at the protein-bonded specific DNA recognition sequence of detected DNA, contain the protein-bonded recognition site of DNA to be measured between be divided into two band toughness ends the half site fragment be probe 1 and probe 2, wherein a double chain oligonucleotide fragment is that probe 1 is fixed in the substrate, and another double chain oligonucleotide fragment is to introduce marker on the probe 2.
This non-marked detects the protein-bonded chip preparation method of DNA, it is characterized in that (1) is according to the protein-bonded specificity binding sequence design of DNA to be detected double chain oligonucleotide probe, being divided into two half site fragment probe 1 and 2, two probes of probe that contain sticky end between the protein-bonded recognition site of DNA can hybridize by sticky end; (2) wherein a double chain oligonucleotide fragment is that probe 1 is fixed in the substrate, and another double chain oligonucleotide fragment is to introduce marker on the probe 2; The perhaps probe 2 any fluorophor of mark not, but with it away from the sticking end that half site 5 ' end is designed to give prominence to, contain some VITAMIN B4 (A) in the base sequence of protruding terminus.
This non-marked detects the protein-bonded chip of DNA, and microballoon, nylon membrane, cellulose acetate membrane, plastics, rubber, pottery or sheet glass etc. are adopted in its probe 1 fixed substrate; The marker of being introduced on its probe 2 is fluorescence molecule group, vitamin H, nano particle etc.
This non-marked detects the double chain oligonucleotide probe that is used for the conjugated protein detection of DNA in the protein-bonded chip of DNA, can be according to the protein-bonded specificity binding sequence of different DNA, design a plurality of immobilization probes that include different albumen recognition sequences, and constitute micro-array chip.
This non-marked detects the protein-bonded chip of DNA, and the sticky end that it is characterized in that probe 1 and probe 2 is that complementary hybridization sequences length is controlled at 2～10 bases, and its sequence is the part in the detected proteic DNA specificity binding sequence.
Non-marked of the present invention detects the application method of the protein-bonded chip of DNA, it is characterized in that (1) joins the probe 2 of mark to contain in the protein-bonded solution of DNA to be detected to carry out preact; (2) with above-mentioned solution and the double chain oligonucleotide immobilization probe or the double chain oligonucleotide microarray hybridization that prepare, under the effect of the conjugated protein specific recognition of DNA, probe 2 specific hybrids be combined in its complementary immobilization probe 1 on; (3) by damping fluid the non-specific adsorption thing of hybridizing on back immobilization probe or the micro-array chip is carried out wash-out, the protein of non-specific binding and probe 2 are eluted from chip; (4) marker on immobilization probe or the micro-array chip behind the wash-out is detected; Perhaps use the archaeal dna polymerase method to establish the Brdurd Triphosaden (dUTP) that mixes mark away from half site 3 ' end, carry out markers tests then at probe 2; When DNA to be measured is conjugated protein when existing, the joint efficiency of probe 1 and probe 2 is very high, can detect the marker of probe 2 on the position of probe 1, otherwise the signal of marker is very weak, realizes thus the protein-bonded non-marked of DNA is detected.
This non-marked detects the application method of the protein-bonded chip of DNA, can adopt the detection method of quantification, conjugated protein with the DNA a certain to be measured of concentration known earlier, set up the typical curve of protein concentration and strength of signal according to above-mentioned detection method, again the concentration of this protein in sample is carried out detection by quantitative.
The present invention compared with prior art has following advantage:
1, great advantage of the present invention is the protein that does not need mark to be detected, has avoided proteinic the losing that may cause in labeling process, and has improved the sensitivity that detects.
2, the present invention is based on a kind of detection method of microarray, can detect simultaneously a large amount of DNA is conjugated protein, and the biological significance of reflection can be saved a large amount of detection costs simultaneously more comprehensively.
3, the present invention has avoided the pollution of the radioactive substance that traditional isotopic labeling brings, and operates safer quick.
4, according to the difference of the marker of introducing, present technique can detect signal in several ways.
Description of drawings
The invention will be further described below with reference to accompanying drawing.
Fig. 1 is that the probe of chip of the present invention prepares synoptic diagram.
Fig. 2 is structural representation Fig. 1 of chip of the present invention.
Fig. 3 is structural representation Fig. 2 of chip of the present invention.
Fig. 4 is a chip application method synoptic diagram 1 of the present invention.
Fig. 5 is a chip application method synoptic diagram 2 of the present invention.
Fig. 6 is the different concns in the chip application method of the present invention and the canonical plotting of strength of signal.
Embodiment
With reference to accompanying drawing 1,2,3, a kind of non-marked detects the protein-bonded chip of DNA, structure has the double chain oligonucleotide probe, the sequence of double chain oligonucleotide probe designs at the protein-bonded specific DNA recognition sequence of detected DNA, contain the protein-bonded recognition site of DNA to be measured between be divided into the half site fragment I of two band toughness ends, II is probe 1 and probe 2 (as Fig. 1), wherein a double chain oligonucleotide fragment is that probe 1 is fixed in the substrate, and another double chain oligonucleotide fragment is to introduce marker (FAM) (as Fig. 2) on the probe 2.The perhaps probe 2 any fluorophor of mark not, but with it away from the sticking end that half site 5 ' end is designed to give prominence to, the base sequence of protruding terminus is 3 '-ANNA-5 ' (wherein N represents any base) (as Fig. 3).1 one kinds of non-marked of embodiment detect the preparation and the application method of the protein-bonded chip of DNA
1, the preparation of probe
According to the protein-bonded specificity binding sequence of DNA to be detected, be divided into two half sites (as Fig. 1) that contain sticky end, two half sites can be hybridized by sticky end.Amino of an end mark of a half site sequence (probe 1) wherein, another half site (probe 2) is marked with fluorescence molecule-FAM (as Fig. 2).
2, the preparation of microarray
According to the probe of step 1 preparation, as shown in Figure 2, (probe 1 NH2) is fixed on surfaces such as nylon membrane, cellulose acetate membrane or sheet glass will to indicate amino.Wherein the corresponding DNA of each probe is conjugated protein.
3, the hybridization of chip
Probe 2 is mixed 25 ℃ hatched 15 minutes with cell extract, then with chip in 25 ℃ of hybridization.Clean the protein (as Fig. 4) of free probe 2 and non-specific binding with elutriant.
4, signal detection
Upward carry out the fluorescent signal detection at optics charge-coupled device (CCD) microscope, Laser Scanning Confocal Microscope or chip fluorescent scanning instrument (as Scanarry Lite) to hybridizing the back chip.
5, the foundation of typical curve
According to step 1～4, with the conjugated protein typical curve (as Fig. 6) of setting up different concns and fluorescent signal of the DNA of concentration known.
6, interpretation of result
According to the power of the fluorescent signal of the corresponding point of testing protein, the combined standard curve is determined the content of this protein in cell.
2 one kinds of non-marked of embodiment detect the preparation and the application method of the protein-bonded chip of DNA
1, the preparation of probe
The design of probe and preparation are basically with example 1, and different is the not any fluorophor of mark of probe 2, but its remote design is become outstanding sticking end, and the base sequence of protruding terminus is 3 '-ANNA-5 ' (wherein N represents any base) (as Fig. 3).
2, the preparation of microarray and hybridization are with example 1.
3, fluorescence molecule mixes
Hybridization back chip is introduced fluorescently-labeled Brdurd Triphosaden (as: Cy3-dUTP) 37 ℃ of effects of archaeal dna polymerase 1 big fragment (Klenow enzyme) extension system 1 hour.(2 * SSC), 0.1% sodium lauryl sulphate (SDS) is cleaned free Cy3-dUTP (as Fig. 5) with 2 times Trisodium Citrates.
4, the detection of fluorescent signal and interpretation of result
The same typical curve (as Fig. 6) that utilizes the method for mixing to set up proteinic concentration of concentration known and fluorescence intensity carries out the detection and the interpretation of result of fluorescent signal with example 1.Example 2 also can come the amplification detection fluorescent signal by the Cy3-dUTP molecule number that increase is mixed, and improves detection sensitivity.
Claims (8)
1, a kind of non-marked detects the protein-bonded chip of DNA, it is characterized in that structure has the double chain oligonucleotide probe, the sequence of double chain oligonucleotide probe designs at the protein-bonded specific DNA recognition sequence of detected DNA, contain the protein-bonded recognition site of DNA to be measured between be divided into two band toughness ends the half site fragment be probe 1 and probe 2, wherein a double chain oligonucleotide fragment is that probe 1 is fixed in the substrate, and another double chain oligonucleotide fragment is to introduce marker on the probe 2.
2, the described a kind of non-marked of claim 1 detects the protein-bonded chip preparation method of DNA, it is characterized in that (1) is according to the protein-bonded specificity binding sequence design of DNA to be detected double chain oligonucleotide probe, being divided into two half site fragment probe 1 and 2, two probes of probe that contain sticky end between the protein-bonded recognition site of DNA can hybridize by sticky end; (2) wherein a double chain oligonucleotide fragment is that probe 1 is fixed in the substrate, and another double chain oligonucleotide fragment is to introduce marker on the probe 2; The perhaps probe 2 any fluorophor of mark not, but with it away from the sticking end that half site 5 ' end is designed to give prominence to, contain some VITAMIN B4 A in the base sequence of protruding terminus.
3, a kind of non-marked according to claim 1 and 2 detects the protein-bonded chip of DNA, it is characterized in that probe 1 fixed substrate employing microballoon, nylon membrane, cellulose acetate membrane, plastics, rubber, pottery or sheet glass.
4, a kind of non-marked according to claim 1 and 2 detects the protein-bonded chip of DNA, it is characterized in that the marker of being introduced on the probe 2 is fluorescence molecule group, vitamin H, nano particle.
5, a kind of non-marked according to claim 1 and 2 detects the protein-bonded chip of DNA, it is characterized in that being used in the chip double chain oligonucleotide probe of the conjugated protein detection of DNA, according to the protein-bonded specificity binding sequence of different DNA, design a plurality of immobilization probes that include different albumen recognition sequences, and constitute micro-array chip.
6, a kind of non-marked according to claim 1 and 2 detects the protein-bonded chip of DNA, the sticky end that it is characterized in that probe 1 and probe 2 is that complementary hybridization sequences length is controlled at 2～10 bases, and its sequence is the part in the detected proteic DNA specificity binding sequence.
7, claim 1 or 2 described a kind of non-marked detect the application method of the protein-bonded chip of DNA, it is characterized in that (1) joins the probe 2 of mark to contain in the protein-bonded solution of DNA to be detected to carry out preact; (2) with above-mentioned solution and the double chain oligonucleotide immobilization probe or the double chain oligonucleotide microarray hybridization that prepare, under the effect of the conjugated protein specific recognition of DNA, probe 2 specific hybrids be combined in its complementary immobilization probe 1 on; (3) by damping fluid the non-specific adsorption thing of hybridizing on back immobilization probe or the micro-array chip is carried out wash-out, the protein of non-specific binding and probe 2 are eluted from chip; (4) marker on immobilization probe or the micro-array chip behind the wash-out is detected; Perhaps use the archaeal dna polymerase method to hold the Brdurd Triphosaden that mixes mark away from half site 3 ', carry out markers tests then at probe 2; When DNA to be measured is conjugated protein when existing, the joint efficiency of probe 1 and probe 2 is very high, can detect the marker of probe 2 on the position of probe 1, otherwise the signal of marker is very weak, realizes thus the protein-bonded non-marked of DNA is detected.
8, a kind of non-marked according to claim 7 detects the application method of the protein-bonded chip of DNA, it is characterized in that adopting the detection method of quantification, conjugated protein with the DNA a certain to be measured of concentration known earlier, set up the typical curve of protein concentration and strength of signal according to above-mentioned detection method, again the concentration of this protein in sample is carried out detection by quantitative.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 03112917 CN1435492A (en) | 2003-03-05 | 2003-03-05 | Chip for non-label detecting DNA bindin, preparation and use method thereof |
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| CN 03112917 CN1435492A (en) | 2003-03-05 | 2003-03-05 | Chip for non-label detecting DNA bindin, preparation and use method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006053463A1 (en) * | 2004-11-18 | 2006-05-26 | Capitalbio Corporation | A testing method of nucleic acid binding protein based on biochip |
| CN100396790C (en) * | 2004-09-17 | 2008-06-25 | 北京大学 | Solution identification and surface addressing protein chip and its preparing and detecting method |
| CN100396791C (en) * | 2004-09-17 | 2008-06-25 | 北京大学 | Method for detecting oligomeric nucleotide through intercalating dye fluorescent resonant energy transfer molecule |
| CN103048463A (en) * | 2012-05-02 | 2013-04-17 | 中国科学院广州生物医药与健康研究院 | Microwell plate nucleic acid hybridization ELISA (enzyme-linked immuno sorbent assay) method for detecting DNA-binding proteins based on different rigors |
| CN115976179A (en) * | 2022-07-12 | 2023-04-18 | 华中农业大学 | High-sensitivity probe and preparation method and application thereof |
-
2003
- 2003-03-05 CN CN 03112917 patent/CN1435492A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100396790C (en) * | 2004-09-17 | 2008-06-25 | 北京大学 | Solution identification and surface addressing protein chip and its preparing and detecting method |
| CN100396791C (en) * | 2004-09-17 | 2008-06-25 | 北京大学 | Method for detecting oligomeric nucleotide through intercalating dye fluorescent resonant energy transfer molecule |
| WO2006053463A1 (en) * | 2004-11-18 | 2006-05-26 | Capitalbio Corporation | A testing method of nucleic acid binding protein based on biochip |
| CN1296492C (en) * | 2004-11-18 | 2007-01-24 | 博奥生物有限公司 | Biochip based method for detecting nucleic acid conjugated protein |
| US8680016B2 (en) | 2004-11-18 | 2014-03-25 | Capitalbio Corporation | Testing method of nucleic acid binding protein based on biochip |
| CN103048463A (en) * | 2012-05-02 | 2013-04-17 | 中国科学院广州生物医药与健康研究院 | Microwell plate nucleic acid hybridization ELISA (enzyme-linked immuno sorbent assay) method for detecting DNA-binding proteins based on different rigors |
| CN115976179A (en) * | 2022-07-12 | 2023-04-18 | 华中农业大学 | High-sensitivity probe and preparation method and application thereof |
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