CN104561290A - Gene chip detection method based on mixed probe - Google Patents
Gene chip detection method based on mixed probe Download PDFInfo
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- CN104561290A CN104561290A CN201410833492.2A CN201410833492A CN104561290A CN 104561290 A CN104561290 A CN 104561290A CN 201410833492 A CN201410833492 A CN 201410833492A CN 104561290 A CN104561290 A CN 104561290A
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- 238000003908 quality control method Methods 0.000 claims abstract description 29
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- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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Abstract
本发明提出了一种基于混合探针基因芯片检测方法，利用两种不同荧光标记对制备的基因芯片有效性进行控制，将目的探针中掺入较短第一荧光基团标记的质量控制探针制备成点样溶液，将点样溶液机械点样于修饰了手臂分子的基片上形成探针斑点，然后将第二荧光基团标记的待测序列与目的探针杂交，检测斑点发出的荧光信号，其中第一荧光基团标记检测目的探针是否固定在芯片基片上，第二荧光基团标记检测待测目的的序列信息。同时使用两种荧光能快速、简便地检测目的探针是否固定在基片上，从而排除因目的探针未固定而出现假阴性的实验结果。
The present invention proposes a gene chip detection method based on mixed probes. Two different fluorescent labels are used to control the effectiveness of the prepared gene chip, and the target probe is mixed with a shorter first fluorescent group-labeled quality control probe. The needle is prepared as a spotting solution, and the spotting solution is mechanically spotted on the substrate modified with arm molecules to form probe spots, and then the sequence to be tested labeled with the second fluorescent group is hybridized with the target probe to detect the fluorescence emitted by the spots signal, wherein the first fluorophore label detects whether the target probe is fixed on the chip substrate, and the second fluorophore label detects sequence information of the target to be detected. Simultaneous use of two kinds of fluorescence can quickly and easily detect whether the target probe is immobilized on the substrate, thereby eliminating false negative experimental results due to unfixed target probe.
Description
Technical field
The present invention relates to gene chip quality controlling means, be specifically related to a kind of based on mixed probe gene chip detection method.
Background technology
How biochip technology, as a fast-developing in recent years biological new and high technology, studies for solving the prospect that function that gene undertakes in vital process provides radiance.Biochip technology refers to and a large amount of probe molecule is fixed on rear on upholder hybridization with the sample molecule of mark, by detecting the hybridization signal intensities of each probe molecule and then obtaining sample sequence information.
Compared with traditional nucleic acid hybridization blot assays, a large amount of feature according to target gene and the pre-designed probe of testing requirement are fixed on support surface by gene chip simultaneously, once hybridization can the relevant information of multiple target gene in determination and analysis sample, the deficiency such as solve traditional nucleic acid blot hybridization technique trivial operations, level of automation is low, operating sequence quantity is few, detection efficiency is low, biochip technology is made to be provided with high-throughput, multiparameter Synchronization Analysis, quick automatical analysis, pinpoint accuracy is analyzed, the feature of high-sensitivity analysis.But with fluorescently-labeled PCR primer and method itself the also existing defects mating probe hybridization and detect unknown DNA sequencing fragment:
1) gene chip fluoroscopic examination depends on the fluorescence intensity of detected result, do not have fluorescence intensity or fluorescence intensity weak be namely judged as feminine gender, but cannot determine whether in the result of feminine gender be probe fixing on; 2) result of study that obtains of several genes chip technology platform is inconsistent, therefore the Validity control of gene chip has become the focus of current gene engineering research, especially applied gene chip there is no some validation checking methods accurately and efficiently, and the popularization of its application is still had any problem.Therefore develop one accurately and efficiently validation checking scheme carry out detection probes and whether fixedly become a kind of exigence.
The Validity control that gene chip has Validity control scheme comprises gene chip probes, prepared by upholder Validity control, probe fixed efficiency on the support and gene chip to prepare, and Validity control during genechip detection, detected result the series of problems such as stdn.This patent mainly for be preparation validity gene chip.
The traditional method of preparation validity gene chip has following several: 1) utilize fluorescent DNA dyes to dye to ssDNA probe, carry out the validity of chip estimate according to fluorescent DNA dyes intensity; 2) utilize object probe 3 sampling liquid and band fluorescence 1 quality control probes 4 sampling liquid respectively point sample in the matrix 7 of having modified arm molecule 6, form respective spot, then make object probe 3 at order spot place and hybridize with DNA sequence dna to be measured 5 that is of the same race or xenogenesis fluorescence 2, whether fluoresce according to quality probe point sample position, the validity of chip estimate, whether fluoresce according to object probe points position, judge DNA sequence dna information to be measured, as shown in Figure 1.But these above methods all cannot evaluate quality prepared by chip directly, effectively.
The method of these traditional gene chip quality controls can decrease the possibility that in gene test, false positive results occurs, improve the accuracy of detected result to a certain extent, but the defect of usual traditional gene chip is himself quality cannot be controlled preferably, the present invention proposes a kind of gene chip Validity control method, can control the validity of gene chip, greatly improve the reliability that it detects.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of based on mixed probe gene chip detection method, can more directly detection probes whether be fixed on substrate, to get rid of the possibility of the negative findings appearance caused because of probe disappearance, improve accuracy and the reliability of genechip detection.
For achieving the above object, the technical solution used in the present invention is:
The present invention is based on mixed probe gene chip detection method, comprise following step:
Step one: sample preparation
The solution of preparation containing object probe, and the solution of the quality control probes of the first fluorophor mark; And the solution of the quality control probes solution of above-mentioned purpose probe and the first fluorophor marked fully mixes, and is prepared into spotting solution.
Step 2: prepared by gene chip
Above-mentioned mixed solution point sample is formed N number of probe spot on the substrate modifying arm molecule, and N is natural number, through fixing and removing loose probe, completes the preparation of gene chip.
Step 3: the signal detection object probe being controlled probe by Detection job is fixed on substrate by arm molecule
Detect the fluorescent signal that above-mentioned all probe spot send, if fluorescence detected under the excitation wavelength of the first fluorophor, then represent that quality control probes is fixed on substrate by arm molecule, illustration purpose probe is fixed on substrate by arm molecule simultaneously, thus judges that gene chip is up-to-standard.
Otherwise, if fluorescence do not detected in any one probe spot under the excitation wavelength of the first fluorophor, represent that quality control probes is not fixed on substrate by arm molecule, be judged to be that gene chip is off quality.
The present invention is based on mixed probe gene chip detection method, further comprising the steps of:
Step 4: hybridization
Hybridized by object probe in probe spot described on the sequence to be measured of the second fluorophor mark and up-to-standard gene chip, after hybridization, the probe in object probe and sequence to be measured combine and form hybrid product.
Step 5: the sequence information determining sequence to be measured
Fluorescent signal detection is carried out to described hybrid product, if fluorescence detected under the excitation wavelength of the second fluorophor, then determines object probe and complementary to be measured, and determine the sequence information of sequence to be measured.
If fluorescence do not detected under the excitation wavelength of the second fluorophor, then represent and can not determine object probe and complementary to be measured, the sequence information of sequence to be measured can not be determined.
Described object probe and quality control probes are thymus nucleic acid, Yeast Nucleic Acid or peptide nucleic acid(PNA).
Described substrate is sheet glass, silica gel wafer, plastics, polypropylene screen, nitrocellulose filter, nylon membrane, miniature magnetic bead or pipe lid.
The first described fluorophor and the second described fluorophor can be Cy3, Cy5, Fitc, FAM or Rhodamine, but the first described fluorophor is not identical when using with the second described fluorophor, and the fluorescence emission peak of two kinds of fluorophors being greater than the difference of 40nm when detecting, preventing both when detecting due to absorption peak overlap and cannot correctly obtain a result.
The first described fluorophor and the second fluorophor are the labeled complex that biotin-avidin is combined with mark substance.
Described mark substance is fluorescin, ferritin, Radioactive colloidal gold, fluorescein or chemiluminescent substance.
The object probe that this patent is carried is one section of known nucleic acid or nucleic acid analogue sequence segment, and has modified active group on nucleic acid or nucleic acid analog chain, is fixed on is modified in the substrate of arm molecule by chemical reaction; Quality control probes is nucleic acid or the nucleic acid analogue sequence segment of one section known, shorter, a terminal modified active group, and be fixed on by chemical reaction and modified in the substrate of arm molecule, the other end marked luminophore or labeled complex; Sequence to be measured is one section of unknown nucleic acid or nucleic acid analogue sequence segment, end mark luminophore or labeled complex; Arm molecule is the small molecular organic compounds of multiple carbon atom composition two ends all containing active group, one end active group can with substrate generation chemical reaction, the other end active group can with the nucleic acid modifying active group or nucleic acid analog chain generation chemical reaction.
Compared with prior art, the beneficial effect that the present invention has is:
1) traditional detection, just certain density quality control probes is fixed on around object probe, quality control probes separates point sample on substrate with object probe, do not consider that the hybridization of object probe and DNA sequence dna to be measured is usually located at solid phase surface, there is steric restriction to a certain degree, if object concentration and probe concentration is too high, object probe is caused to fix not get on, and the present invention fixes after quality control probes and object probe fully being mixed again, for the purpose of probe and DNA sequence dna to be measured enough hybridization locus are provided, greatly facilitate DNA sequence dna to be detected and object probe hybridization like this.
2) quality control probes that the object of the invention probe and shorter fluorophor mark mixes, because object probe, quality control probes have identical active group, common Chemical bond mode is had again with arm molecule, be fixed on substrate, so by detection first fluorophor, that determines quality control probes is fixed on substrate, indirectly can determine that object probe is fixed on substrate, ensure that the quality of chip manufacturing, this mode also reaches the validity of the whole chip of Real-Time Monitoring.
3) the present invention is by detection first fluorophor, determine the quality of gene chip, provide reliable negative decision method, this negative findings, can not change because of probe fixed efficiency, because when probe fixed efficiency is low, the signal of the first fluorophor very lowly even can not have signal, also can get rid of false positive misjudgement in gene chip hybridization interpretation simultaneously, and existence is detected that hybridization signal is weak and to detect hybridization signal strong, also can obtain true negative result, thus solve because gene chip quality problems are to experimental result interference problem.
4) invention is a kind of based on mixed probe gene chip detection method, is applied to accuracy and the reliability that greatly can to improve gene test result in gene chip preparation.Simultaneously, not only the method for quality control can be provided to improve the quality of production for the producer of gene chip, evaluation can be made to chip before the use again for the user of gene chip, find possibility produced problem in experiment simply and effectively, thus the analytic process decreased in experiment, improve conventional efficient.
Accompanying drawing explanation
Fig. 1 is the structural representation of the method detecting gene chip validity in prior art;
Fig. 2 is the one-piece construction schematic diagram that the present invention is based on mixed probe gene chip detection method;
Fig. 3 is the partial enlargement structural representation that the present invention is based on mixed probe gene chip detection method.
In figure: 1, the first fluorophor, the 2, second fluorophor, 3, object probe, 4, quality control probes, 5, DNA sequence dna to be measured, 6, arm molecule, 7, substrate.
Embodiment
Below in conjunction with accompanying drawing, the invention will be further described.
The present invention is based on mixed probe gene chip detection method, comprise following step:
1, sample preparation
As shown in Figure 2,3, the solution of preparation containing object probe 3, and the liquid of the quality control probes 4 of the first fluorophor 1 mark; And the spotting solution of the quality control probes 4 solution of above-mentioned purpose probe 3 and the first fluorophor mark 1 marked fully mixes, and is prepared into spotting solution.
2, gene chip preparation
Above-mentioned mixed solution point sample is formed N number of probe spot on the substrate 7 having modified arm molecule 6, and N is natural number, through fixing and removing loose probe, completes the preparation of gene chip.
3, be fixed on substrate 7 by arm molecule 6 by the signal detection object probe 3 of Detection job control probe 4
Detect the fluorescent signal that above-mentioned all probe spot send, if fluorescence detected under the excitation wavelength of the first fluorophor 1, then represent that quality control probes 4 is fixed on substrate 7 by arm molecule 6, illustration purpose probe 3 is fixed on substrate 7 by arm molecule 6 simultaneously, thus judges that gene chip is up-to-standard.
Otherwise, if fluorescence do not detected in any one probe spot under the excitation wavelength of the first fluorophor 1, represent that quality control probes 4 is not fixed on substrate 7 by arm molecule 6, be judged to be that gene chip is off quality.
4, hybridize:
The sequence to be measured 5 marked by second fluorophor 2 is hybridized with the object probe 3 in the probe spot of point sample on up-to-standard gene chip, and after hybridization, the probe in object probe 3 and sequence to be measured 5 combine and form hybrid product.
5, the sequence information of sequence 5 to be measured is determined:
Fluorescent signal detection is carried out to described hybrid product, if fluorescence detected under the excitation wavelength of the second fluorophor 2, then determines that object probe 3 is complementary with sequence 5 to be measured, and determine the sequence information of sequence 5 to be measured.
If fluorescence do not detected under the excitation wavelength of the second fluorophor 2, then can not determine that object probe 3 is complementary with sequence 5 to be measured, the sequence information of sequence 5 to be measured cannot be determined.
By the quality control probes 4 that the shorter fluorophor 1 being mixed into higher concentration in object probe 3 marks, advantage can provide locus for original object probe 3 and sequence to be measured, facilitate sequence 5 to be measured to be combined with object probe 3, and be combined in same modification substrate with corresponding object probe 3 respectively by multiple sequence 5 to be measured and form a kind of gene chip that may be used for Mass Control.
By checking under the excitation wavelength of fluorophor 1 whether probe is fixed on substrate, then under the excitation wavelength of fluorophor 2, detect fragment sequence 5 to be measured; If fluorescence do not detected under the excitation wavelength of fluorophor 1, represent that probe is not fixed, if at this moment can detect, fluorescence does not detect fluorescence under the excitation wavelength of fluorophor 2, shows that result is negative, if twice all detects fluorescence, result is positive, explanation chip quality is qualified, sequence 5 to be measured is complementary with object probe 3, judges the sequence information of sequence to be measured further.
Embodiment 1: genetically engineered soybean detects gene chip
1, sample preparation
By the object probe 5'-NH of genetically engineered soybean
2the solution of-CTG AAG GCG GGA AAC GAC AAT CTG-3', and adopt Cy5 to control probe 5'-NH as the first fluorophor 1 mark quality
2above-mentioned two spotting solutions are fully mixed, are prepared into spotting solution by the solution of-TTTTTTTTTTTTTTT-3'.
2, gene chip preparation
By above-mentioned mixed spotting solution point sample on aldehyde radical sheet, aldehyde radical sheet is the substrate being prepared into aldehyde radical end modified through the sheet glass of silane, glutaraldehyde process, aldehyde radical sheet forms N number of probe spot, N is natural number, through fixing and removing loose probe, complete the preparation of gene chip.
3, probe 5'-NH is controlled by Detection job
2the object probe 5'-NH of the signal detection genetically engineered soybean of-TTTTTTTTTTTTTTT-3'
2-CTG AAG GCG GGA AAC GAC AAT CTG-3' is fixing on the glass sheet by aldehyde radical
Detect the fluorescent signal that above-mentioned all probe spot send, if fluorescence detected under excitation wavelength 633 nm of Cy5, then represent quality control probes 5'-NH
2-TTTTTTTTTTTTTTT-3' is fixing on the glass sheet by aldehyde radical, and the object probe 5'-NH of genetically engineered soybean is described simultaneously
2-CTG AAG GCG GGA AAC GAC AAT CTG-3' can be fixing on the glass sheet by aldehyde radical, thus judge that gene chip is up-to-standard.
Otherwise, if fluorescence do not detected in any one probe spot under excitation wavelength 633 nm of Cy5, represent quality control probes 5'-NH
2-TTTTTTTTTTTTTTT-3' is unfixing on the glass sheet by aldehyde radical, and chip quality is defective.
4, hybridize:
By upstream primer 5'-CTG CTC CAC TCT TCC TTT-3', downstream primer 5'-AGA CTC TGT ACC CTG ACC T-3', and transgenic soybean gene group, under the effect of Tak enzyme, the sequence to be measured 5 of Cy3 mark is obtained, the object probe 5'-NH of the probe spot genetically engineered soybean of point sample on the sequence to be measured marked by above-mentioned Cy3 and up-to-standard gene chip through pcr amplification
2-CTG AAG GCG GGA AAC GAC AAT CTG-3' is hybridized, after hybridization, and the object probe 5'-NH of genetically engineered soybean
2-CTG AAG GCG GGA AAC GAC AAT CTG-3' and sequence to be measured combine and form hybrid product.
5, the sequence information of sequence 5 to be measured is determined
Fluorescent signal detection is carried out to hybrid product, if fluorescence detected under excitation wavelength 533 nm of Cy3, then determines the object probe 5'-NH of genetically engineered soybean
2-CTG AAG GCG GGA AAC GAC AAT CTG-3' and sequence to be measured 5 complementation, and determine sequence 5 to be measured containing sequence 5'-CAG ATT GTC CTT ACC CGC GTT CAG-3'.
If fluorescence do not detected under excitation wavelength 533 nm of Cy3, then represent the object probe 5'-NH of genetically engineered soybean
2-CTG AAG GCG GGA AAC GAC AAT CTG-3' is unfixing on the glass sheet by aldehyde radical, can not determine the object probe 5'-NH of genetically engineered soybean
2-CTG AAG GCG GGA AAC GAC AAT CTG-3' and sequence to be measured 5 complementation, thus the arrangement information of sequence 5 to be measured cannot be determined.
Claims (8)
1. based on a mixed probe gene chip detection method, it is characterized in that, the method comprises following step:
Step one: sample preparation
Preparation is containing the solution of object probe (3), and the solution of quality control probes (4) that the first fluorophor (1) marks; And the spotting solution of the quality control probes (4) solution of above-mentioned purpose probe (3) and the first fluorophor mark (1) marked fully mixes, and is prepared into spotting solution;
Step 2: prepared by gene chip
Above-mentioned mixed solution point sample is formed N number of probe spot in the substrate (7) having modified arm molecule (6) is upper, and N is natural number, through fixing and removing loose probe, completes the preparation of gene chip;
Step 3: the signal detection object probe (3) being controlled probe (4) by Detection job is fixed on substrate (7) by arm molecule (6);
Detect the fluorescent signal that above-mentioned all probe spot send, if fluorescence detected under the excitation wavelength of the first fluorophor (1), then represent that quality control probes (4) is fixed on substrate (7) by arm molecule (6), illustration purpose probe (3) is fixed on substrate (7) by arm molecule (6) simultaneously;
Otherwise, if fluorescence do not detected in any one probe spot under the excitation wavelength of the first fluorophor (1), represent that quality control probes (4) is not fixed on substrate (7) by arm molecule (6).
2. according to claim 1ly to it is characterized in that based on mixed probe gene chip detection method, further comprising the steps of:
Step 4: hybridization
Object probe (3) in probe spot described on the sequence to be measured (5) marked by second fluorophor (2) and up-to-standard gene chip is hybridized, and after hybridization, the probe in object probe (3) and sequence to be measured (5) combine and form hybrid product;
Step 5: the sequence information determining sequence to be measured (5)
Fluorescent signal detection is carried out to described hybrid product, if fluorescence detected under the excitation wavelength of the second fluorophor (2), then determines that object probe (3) is complementary with sequence to be measured (5), and determine the sequence information of sequence to be measured (5);
If fluorescence do not detected under the excitation wavelength of the second fluorophor (2), then represent and can not determine that object probe (3) is complementary with sequence to be measured (5), the sequence information of sequence to be measured (5) cannot be determined.
3. according to claim 2ly to it is characterized in that based on mixed probe gene chip detection method, described object probe (3) and quality control probes (4) they be thymus nucleic acid, Yeast Nucleic Acid or peptide nucleic acid(PNA).
4. according to claim 2ly to it is characterized in that based on mixed probe gene chip detection method, described substrate (7) be sheet glass, silica gel wafer, plastics, polypropylene screen, nitrocellulose filter, nylon membrane, miniature magnetic bead or pipe lid.
5. according to claim 2ly it is characterized in that based on mixed probe gene chip detection method, described the first fluorophor (1) is not identical with described the second fluorophor (2), and the fluorescence emission peak of above-mentioned two kinds of fluorophors is greater than 40nm.
6. according to claim 5ly it is characterized in that based on mixed probe gene chip detection method, described the first fluorophor (1) and described the second fluorophor (2) are Cy3, Cy5, Fitc, FAM or Rhodamine.
7. according to claim 5ly to it is characterized in that based on mixed probe gene chip detection method, the labeled complex that described the first fluorophor (1) and the second fluorophor (2) are combined with mark substance for biotin-avidin.
8. according to claim 7 based on mixed probe gene chip detection method, it is characterized in that described mark substance is fluorescin, ferritin, Radioactive colloidal gold, fluorescein or chemiluminescent substance.
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| CN201410833492.2A CN104561290A (en) | 2014-12-30 | 2014-12-30 | Gene chip detection method based on mixed probe |
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| CN201410833492.2A CN104561290A (en) | 2014-12-30 | 2014-12-30 | Gene chip detection method based on mixed probe |
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| CN108138225A (en) * | 2015-07-27 | 2018-06-08 | 亿明达股份有限公司 | The space orientation of nucleic acid sequence information |
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