CN104561292A - Gene chip detection method based on temperature difference probes - Google Patents

Abstract

The present invention proposes a probe gene chip detection method based on temperature difference, using two different fluorescent markers to control the quality of the gene chip. Perform hybridization first, detect the first fluorescent group, and judge whether the target probe is immobilized on the substrate; at a higher temperature, the target probe is hybridized with the DNA sequence to be detected labeled with the second fluorescent group, and the second fluorescent group is detected. Determine the detected DNA sequence information. Using two kinds of fluorescence at the same time can quickly and easily detect whether the target probe is immobilized on the substrate, thereby eliminating false negative results due to unfixed target probes and improving the accuracy and reliability of genetic testing results.

Description

A gene chip detection method based on temperature difference probe

Technical field:

The invention belongs to the field of gene chip quality control, in particular to a gene chip detection method based on temperature difference probes.

Background technique:

As a biological high-tech developed rapidly in recent years, gene chip technology provides a bright prospect for how to study the functions of genes in the course of life. Gene chip technology refers to immobilizing a large number of probe molecules on the support and hybridizing with labeled sample molecules, and then obtaining the number and sequence information of sample molecules by detecting the hybridization signal intensity of each probe molecule.

Compared with the traditional nucleic acid blot hybridization method, the gene chip immobilizes a large number of pre-designed probes on the surface of the support according to the characteristics and detection requirements of the target gene. One hybridization can detect and analyze the relevant information of multiple target genes in the sample. It solves the shortcomings of traditional nucleic acid imprinting hybridization technology such as complicated operation, low degree of automation, small number of operation sequences, and low detection efficiency. Features of high sensitivity analysis. However, the method of using fluorescently labeled PCR products to hybridize with matching probes to detect unknown DNA sequence fragments also has defects:

1) Gene chip fluorescence detection mainly depends on the fluorescence intensity of the test result. If there is no fluorescence intensity or the fluorescence intensity is weak, it is judged as negative, but it is impossible to determine whether the probe is immobilized in the negative result; 2) Multiple gene chip technologies The research results obtained by the platform are inconsistent, so the effectiveness control of gene chips has become a hot spot in gene technology research. In particular, there are no accurate and efficient effectiveness detection methods for applied gene chips, and its application is still difficult to promote. Therefore, it is an urgent need to develop an accurate and efficient effectiveness detection scheme to detect whether the probe is immobilized.

The effectiveness control scheme of the gene chip includes the effectiveness control of gene chip probes and support preparation, the immobilization efficiency of probes on the support, which is the effectiveness control of gene chip preparation, and the effectiveness control and detection of gene chip detection. The standardization of results and other issues. This patent is mainly aimed at preparing effective gene chips.

There are several traditional methods for preparing effective gene chips: one method is to use fluorescent DNA dyes to stain single-stranded DNA probes, and estimate the effectiveness of the chips according to the intensity of fluorescent DNA dyes; The quality control probe 8 labeled with the fluorescent group 1 is spotted on the substrate 7 modified with the arm molecule 6 to form respective spots, and then the target probe 3 has the same or the same species as the band group at the spots. Hybridization of the DNA sequence to be tested marked with heterogeneous fluorophore 2, the effectiveness of the chip is estimated according to whether the spot position of the quality probe 8 emits fluorescence, and the information of the DNA sequence to be tested is determined according to whether the spot position of the target probe fluoresces, as shown in the figure 1 shown. However, none of the above methods can directly and effectively evaluate the quality of chip preparation.

This method of gene chip quality control can reduce the possibility of false positive results in genetic testing and greatly improve the accuracy of testing results. Usually the defect of the gene chip is that its own quality cannot be well controlled. If there is a method that can control the effectiveness of such a new type of gene chip to the chip, then the reliability of its detection will be greatly improved. The promotion of its use goes one step further.

Invention content:

Aiming at the deficiencies of the prior art, the present invention provides a probe gene chip detection method based on temperature difference, which can more directly detect whether the probe is fixed on the substrate, so as to eliminate the occurrence of negative results caused by the absence of the probe. Possibility, improve the accuracy and reliability of gene chip detection.

In order to achieve the above object, the technical scheme adopted in the present invention is:

The present invention is based on temperature difference probe gene chip detection method and comprises the following steps:

Step 1: Sample preparation:

Prepare a solution containing the target probe and a solution of the oligomer sequence labeled with the first fluorophore; fully mix the solution of the above-mentioned target probe and the solution of the oligomer sequence labeled with the first fluorophore, and prepare a dot sample solution;

Step 2: Gene chip preparation

Spotting the above-mentioned mixed spotting solution on the substrate modified with arm molecules to form N probe spots, N is a natural number, after fixing, at low temperature _T1 , the range of low temperature _T1 is 0 ~ 30 ° C, so In all the above-mentioned probe spots, the target probe is hybridized with the oligomer sequence labeled with the first fluorescent group, and the first hybridization product solution corresponding to each probe spot is obtained; unfixed probes and unhybridized oligos are removed. Polymer sequence to complete the preparation of the gene chip;

Step 3: Detect the target probe by detecting the signal of the oligomer sequence (4) and immobilize it on the substrate through the arm molecule:

Detect the fluorescent signals emitted by all the first hybridization product solutions above. If fluorescence is detected at the excitation wavelength of the first fluorescent group, it means that the oligomer sequence is immobilized on the substrate through the arm molecule, and at the same time, it indicates that the target probe is passed through the arm molecule. Fixed on the substrate, so as to determine the quality of the gene chip;

Otherwise, if no fluorescence is detected at the excitation wavelength of the first fluorescent group in any spot, it means that the oligomer sequence is not immobilized on the substrate by the arm molecule, and the quality of the gene chip is determined to be unqualified.

The method for detecting the effectiveness of the gene chip by the hybrid probe of the present invention also includes the following steps:

Step 4: Hybridization

The sequence to be tested labeled with the second fluorophore is hybridized with the target probe in the spots spotted on the qualified gene chip at a high temperature T ₂ , and the high temperature T ₂ ranges from 20 to 60°C. After hybridization, the target The probe in the probe combines with the sequence to be detected to form a second hybridization product;

Step 5: Determine the sequence information of the sequence to be tested

Perform fluorescence signal detection on the second hybridization product, if fluorescence is detected at the excitation wavelength of the second fluorophore, then determine that the target probe is complementary to the sequence to be tested, and determine the sequence information of the sequence to be tested;

If no fluorescence is detected at the excitation wavelength of the second fluorophore, it means that it cannot be determined that the target probe is complementary to the sequence to be detected, and the sequence information of the sequence to be detected cannot be determined.

The difference between said low temperature and high temperature is not less than 10°C.

The target probe is deoxyribonucleic acid, ribonucleic acid or peptide nucleic acid.

The substrate is glass sheet, silica gel wafer, plastic, polypropylene film, nitrocellulose film, nylon film, miniature magnetic beads or tube cover.

The first fluorescent group and the second fluorescent group can be Cy3, Cy5, Fitc, FAM or Rhodamine, but the first fluorescent group and the second fluorescent group are used without The same, and the difference between the fluorescence emission peaks of the two fluorophores should be greater than 40nm during detection, so as to prevent the two from being unable to obtain correct results due to overlapping absorption peaks during detection.

The first fluorescent group and the second fluorescent group are labeling complexes in which biotin-avidin is combined with a labeling substance.

The labeling substance is fluorescent protein, ferritin, colloidal gold, fluorescein or chemiluminescent substance.

The target probe mentioned in this patent is a known nucleic acid or nucleic acid analog sequence fragment, and the active group is modified on the nucleic acid or nucleic acid analog chain, which can be fixed on the substrate of the modified arm molecule through a chemical reaction; The oligomer sequence is a known, short nucleic acid or nucleic acid analog sequence fragment, the end is labeled with a luminescent group or a labeling complex; the test sequence is an unknown nucleic acid or nucleic acid analog sequence fragment, and the end is labeled with a luminescence Group or labeling complex; the arm molecule is a small molecular organic compound composed of multiple carbon atoms with active groups at both ends. The active group at one end can chemically react with the substrate, and the active group at the other end can interact with the modified active group. A chemical reaction of the nucleic acid or nucleic acid analog strands of the group.

Compared with prior art, the beneficial effect that the present invention has is:

1) In the traditional detection, only a certain concentration of quality control probe is fixed around the target probe, and the quality control probe and the target probe are separately spotted on the substrate, without considering the relationship between the target probe and the DNA sequence to be tested. Hybridization is usually located on the surface of the solid phase, and there is a certain degree of steric hindrance. If the concentration of the target probe is too high, the target probe cannot be immobilized. However, in the present invention, the target probe and the first fluorophore-labeled oligomer Sequence hybridization provides hybridization spatial position for the subsequent target probe and the DNA sequence to be tested, which greatly facilitates the hybridization of the DNA sequence to be detected and the target probe.

2) The target probe is mixed with the oligomer sequence labeled with the first fluorescence, sampled, and hybridized. Because the target probe and the oligomer sequence are complementary, there is a force between molecules. By detecting the first fluorescence, the target can be determined Whether the probe is bound to the substrate through the arm molecules ensures the quality of the chip production, and this method also achieves real-time monitoring and real-time monitoring of the effectiveness of the entire chip.

3) Because the oligomer sequence is shorter than the target probe, at a low temperature of 0-30 °C, the target probe hybridizes with the oligomer sequence labeled with the first fluorescence, and at a high temperature of 20-60 °C, the target probe Hybridization with the sequence to be detected labeled with the second fluorophore, through the control of the hybridization temperature to achieve the type of hybridization with the target probe and the oligomer sequence or the sequence to be detected, the design is exquisite and the operation is simple.

4) The method for detecting the validity of the gene chip of the present invention, if applied to the preparation of the gene chip, can greatly improve the accuracy and reliability of the gene detection results; at the same time, it can not only provide the method of effectiveness control for the manufacturer of the gene chip , improve production quality, and can evaluate the chip for the user of the gene chip before use, and find out possible problems in the experiment simply and effectively, thereby reducing the analysis process in the experiment and improving the efficiency of the experiment.

Description of drawings

Fig. 1 is a structural schematic diagram of a method for the effectiveness of a gene chip in the prior art;

Fig. 2(a) is a schematic diagram of the structure of the gene chip detection method based on temperature difference probes at low temperature according to the present invention;

Fig. 2(b) is a schematic diagram of the structure of the gene chip detection method based on temperature difference probes at high temperature according to the present invention;

Figure 3 (a) is a schematic diagram of a partial enlarged structure of the gene chip detection method based on temperature difference probes at low temperature in the present invention;

Fig. 3(b) is a schematic diagram showing a partial enlarged structure of the gene chip detection method based on the temperature difference probe of the present invention at high temperature.

In the figure: 1. First fluorescent group, 2. Second fluorescent group, 3. Target probe, 4. Oligomer sequence, 5. Sequence to be tested, 6. Arm molecule, 7. Substrate, 8. Quality control probes.

Detailed ways

The present invention will be further described below in conjunction with accompanying drawing.

The present invention is based on temperature difference probe gene chip detection method, comprises the following steps:

1. Sample preparation:

As shown in Figure 2 a, 2b, 3a, 3b, prepare the solution that contains the solution of purpose probe 3 and the oligomer sequence 4 of the first fluorescent group 1 label; And the solution of above-mentioned purpose probe 3 and the first The solution of oligomer sequence 4 labeled with fluorophore label 1 is fully mixed to prepare a spotting solution;

2. Gene chip preparation

Spotting the above mixed spotting solution on the substrate 7 modified with the arm molecule 6 to form N probe spots, where N is a natural number, fixed, at a low temperature _T1 , the low temperature _T1 ranges from 0 to 30 ° C, the target probe 3 in all the probe spots is hybridized with the oligomer sequence 4 labeled with the first fluorescent group 1, and the first hybridization product solution corresponding to each probe spot is obtained; unfixed probes are removed Needle 3 and unhybridized oligomer sequence 4 to complete the preparation of the gene chip;

3. By detecting the signal of the oligomer sequence 4, the target probe 3 is immobilized on the substrate 7 through the arm molecule 6:

Detect the fluorescent signals emitted by all the first hybridization product solutions above. If fluorescence is detected at the excitation wavelength of the first fluorescent group 1, it means that the oligomer sequence 4 is immobilized on the substrate 7 through the arm molecule 6, and the purpose is explained at the same time. The probe 3 is fixed on the substrate 7 through the arm molecule 6, so as to judge the quality of the gene chip;

Otherwise, if no fluorescence is detected in the first hybridization product solution corresponding to any probe spot at the excitation wavelength of the first fluorescent group 1, it means that the oligomer sequence 4 is not immobilized on the substrate 7 through the arm molecule 6, and it is determined that The quality of the gene chip is unqualified.

4. Hybridize the sequence to be tested

The sequence 5 to be tested labeled with the second fluorophore is hybridized with the target probe 3 in the probe spot described on the quality-qualified gene chip at a high temperature T ₂ , the high temperature T ₂ ranges from 20 to 60°C, and the hybridization Finally, the probe in the target probe 3 is combined with the test sequence 5 to form a second hybridization product;

5. Determine the sequence information of the sequence 5 to be tested

Perform fluorescence signal detection on the second hybridization product, if fluorescence is detected at the excitation wavelength of the second fluorophore 1, then determine that the target probe 3 is complementary to the sequence to be detected 5, and determine the sequence information of the sequence to be detected 5 ;

If no fluorescence is detected at the excitation wavelength of the second fluorophore, it means that the complementarity between the target probe 3 and the test sequence 5 cannot be determined, and the sequence information of the test sequence 5 cannot be determined.

In the present invention, by first hybridizing the target probe with the oligomer sequence labeled with the first fluorescent group 1, the advantage is that it can provide a spatial position for the target probe and the sequence to be tested, facilitate the combination of the sequence to be tested and the target probe, and A gene chip that can be used for quality control is formed by combining multiple sequences to be tested with corresponding target probes in the same modified substrate.

By checking whether the probe is fixed on the substrate under the excitation wavelength of the first fluorescent group, and then detecting the DNA sequence of the fragment to be tested under the excitation wavelength of the first fluorescent group. If no fluorescence is detected at the excitation wavelength of the second fluorophore, it means that the probe is not immobilized. If fluorescence can be detected at this time but no fluorescence is detected at the excitation wavelength of the first fluorophore, it indicates that the result is negative. If fluorescence is detected both times, the result is positive. The difference is that the temperature for stable hybridization of the target probe with the oligomer sequence labeled with the first fluorophore is lower at 0-30 °C, while the temperature for stable hybridization with the sample is higher at 20-60 °C. That is to say, at a lower temperature of 0-30 °C, the oligomer sequence labeled with the first fluorescent group can hybridize stably with the target probe (as shown in Figure 2a), while at a higher temperature of 20-60 °C, The target probe can stably hybridize with the sequence to be tested (as shown in Figure 2b). Therefore, the fluorescence emitted by the second fluorophore should be detected before the probe of interest is hybridized to the sample.

Example 1: Transgenic soybean detection gene chip

1. Sample preparation:

Prepare the solution of the target probe 5'-NH ₂ -AAA AAA AAA AAA AAA CTG AAC GGG AAA CGA CAA TCT G-3' of transgenic soybean, and use the first fluorescent group Cy5 to label the oligomer sequence 5'-Cy5- For the solution of TTT TTT TTT TTT TTT-3', thoroughly mix the above two spotting solutions to prepare a spotting solution.

2. Gene chip preparation:

The above-mentioned mixed spotting solution is spotted on an aldehyde-based sheet, and the aldehyde-based sheet is prepared from a glass sheet treated with silane and glutaraldehyde to form a substrate with an aldehyde group modified at the end, and N probe spots are formed on the aldehyde-based sheet. N is a natural number. After fixing, at a low temperature of 25°C, the target probe 5'-NH ₂ -AAA AAA AAA AAA AAA CTG AAG GCG GGA AAC GAC AAT CTG-3' and Cy5-labeled oligo The polymer sequence 5'-Cy5-TTT TTT TTT TTT TTT-3' undergoes a hybridization reaction to obtain the first hybridization product solution corresponding to each probe spot; remove unfixed probe 3 and unhybridized oligomer sequence 4 , to complete the preparation of the gene chip.

3. Detect the target probe 5'-NH ₂ -CTG AAG GCG GGA AAC GAC AAT CTG-3' of transgenic soybean by detecting the signal of the oligomer sequence 5'-Cy5-TTT TTT TTT TTT TTT TTT-3' Aldehyde groups immobilized on glass slides

Detect the fluorescence signals from all the first hybridization product solutions above. If fluorescence is detected at the excitation wavelength of Cy5 at 633 nm, it means that the solution of the oligomer sequence 5'-Cy5-TTT TTT TTT TTT TTT-3' passed through the aldehyde group Immobilized on the glass slide, and at the same time, it shows that the target probe of transgenic soybean 5'-NH ₂ -AAA AAA AAA AAA AAA CTG AAG GCG GGA AAC GAC AAT CTG-3' can be fixed on the glass slide through the aldehyde group, so as to determine the quality of the gene chip .

Otherwise, if no fluorescence is detected in the solution of the first hybridization product corresponding to any probe spot at the excitation wavelength of Cy5 at 633 nm, it means that the solution of the oligomer sequence 5'-Cy5-TTT TTT TTT TTT TTT-3' failed to If the aldehyde group is fixed on the glass slide, it is judged that the quality of the gene chip is unqualified.

4. Hybridize the DNA sequence to be tested:

Under the action of the upstream primer 5'-CTG CTC CAC TCT TCC TTT-3', the downstream primer 5'-AGA CTC TGT ACC CTG ACC T-3', and the transgenic soybean genome and Tak enzyme, the second fluorescence was amplified by PCR Group Cy3-labeled test sequence 5, combine it with the target probe 5'-NH ₂ -AAA AAA AAA AAA AAA CTG AAG GCG GGA AAC GAC AAT CTG-3 in the probe spots described on the qualified gene chip 'Hybridize at 55 °C. After hybridization, the probe in the target probe 5'-NH ₂ -AAA AAA AAA AAA AAA CTG AAG GCG GGA AAC GAC AAT CTG-3' combines with the test sequence 5 to form a second hybridization product.

5. Determine the sequence information of the sequence 5 to be tested:

Fluorescence signal detection is performed on the second hybridization product solution, and if fluorescence is detected at an excitation wavelength of Cy3 of 533 nm, then the target probe 5'-NH ₂ -AAA AAA AAA AAA AAA CTG AAG GCG GGA AAC of the transgenic soybean is determined GAC AAT CTG-3' is complementary to the sequence to be tested, and it is determined that the sequence information of the sequence to be tested 5 contains the sequence 5'-CAG ATT GTC CTT ACC CGC GTT CAG-3'.

If no fluorescence is detected at the excitation wavelength of Cy3 at 533 nm, it means that the target probe 5'-NH ₂ -AAA AAA AAA AAA AAA CTG AAG GCG GGA AAC GAC AAT CTG-3' and the sequence to be tested cannot be determined for transgenic soybeans 5 complementary, so that the arrangement information of the sequence to be tested cannot be determined.

Claims (9)

1., based on a temperature head opposite sex probe gene chip detection method, it is characterized in that, the method comprises following step:

Step one: sample preparation:

The solution of the oligomer sequence (4) that preparation marks containing the solution of object probe (3) and the first fluorophor (1); And the solution of the oligomer sequence (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

By above-mentioned mixed spotting solution point sample, in modifying, the substrate (7) in arm molecule (6) is upper forms N number of probe spot, and N is natural number, through fixing, at low temperature T ₁under, low temperature T ₁scope is 0 ~ 30 ° of C, and hybridization occurs the oligomer sequence (4) that in described all probe spot, object probe (3) and the first fluorophor (1) mark, and obtains the first hybrid product solution that each probe spot is corresponding; The oligomer sequence removed loose probe and do not hybridize, completes the preparation of gene chip;

Step 3: be fixed on substrate (7) by the signal detection object probe (3) detecting oligomer sequence (4) by arm molecule (6) upper:

Detect the fluorescent signal that above-mentioned all first hybrid product solution sends, if fluorescence detected under the excitation wavelength of the first fluorophor (1), then represent that oligomer sequence (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 the first hybrid product solution corresponding to any one probe spot does not detect fluorescence under the excitation wavelength of the first fluorophor (1), represent that oligomer sequence (4) is not fixed on substrate (7) by arm molecule (6).

2. according to claim 1ly to it is characterized in that based on temperature head opposite sex probe gene chip detection method, further comprising the steps of:

Step 4: sequence to be measured (5) is hybridized

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 at high temperature T ₂under hybridize, high temperature T ₂scope is 20 ~ 60 ° of C, and after hybridization, the probe in object probe (3) and sequence to be measured (5) are in conjunction with formation second hybrid product;

Step 5: the sequence information determining sequence to be measured (5)

Fluorescent signal detection is carried out to described second 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 2 based on temperature head opposite sex probe gene chip detection method, it is characterized in that, described low temperature and high temperature differ and are not less than 10 ° of C.

4. according to claim 2 based on temperature head opposite sex probe gene chip detection method, it is characterized in that, described object probe (3) is thymus nucleic acid, Yeast Nucleic Acid or peptide nucleic acid(PNA).

5. according to claim 2 based on temperature head opposite sex probe gene chip detection method, it is characterized in that, described substrate (7) is sheet glass, silica gel wafer, plastics, polypropylene screen, nitrocellulose filter, nylon membrane, miniature magnetic bead or pipe lid.

6. according to claim 2 based on temperature head opposite sex probe gene chip detection method, it is characterized in that, 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.

7. according to claim 6 based on temperature head opposite sex probe gene chip detection method, it is characterized in that, described the first fluorophor (1) and described the second fluorophor (2) are Cy3, Cy5, Fitc, FAM or Rhodamine.

8. according to claim 6 based on temperature head opposite sex probe gene chip detection method, it is characterized in that, the labeled complex that described the first fluorophor (1) and the second fluorophor (2) are combined with mark substance for biotin-avidin.

9. according to claim 8 based on temperature head opposite sex probe gene chip detection method, it is characterized in that described mark substance is fluorescin, ferritin, Radioactive colloidal gold, fluorescein or chemiluminescent substance.