Gene chip technology
Summary
Gene chips, also known as DNA chips, DNA array, etc., refers to the in situ synthesis of oligonucleotides on a solid-phase support or the direct curing of a large number of DNA probes on the surface of the support in an orderly manner by microprinting.
Then hybridize with the labeled samples, and realize rapid, parallel and efficient detection of DNA and other biological samples by detecting hybridization signals.
It is a high and new technology which is a cross-fusion of microelectronics, computer, molecular biology and other disciplines, and has a wide range of applications in the fields of DNA sequencing, gene expression analysis, genome research, gene diagnosis, drug research and development, as well as industrial and agricultural, food and environmental monitoring.
Principle
Gene chips can be categorized into two main types according to their preparation: synthetic gene chips and DNA microarrays.
The basic principle of synthetic gene chip is to synthesize oligonucleotides in situ in specific parts of the chip by using techniques such as photolithography.
This type of chip has a higher degree of integration, but the length of the synthesized oligonucleotide probe is shorter, generally 8 to 20 bases, and the longest length does not exceed 50 bases.
Therefore, for genes of average length, it is necessary to use multiple probes that overlap each other for accurate identification of the gene.
Although the physical integration is high, the integration of biogenetic information is relatively compromised. Moreover, the preparation of such microarrays is strictly controlled by patents, and the development is limited.
The basic principle of DNA microcollection arrays is to use a set of special chip-printing devices to sequentially solidify pre-prepared gene probes on the surface of the support by microprinting through the back-and-forth movement of a robotic arm.
Although the integration degree of the chip is relatively low, but the use of the probe group source is more flexible, but the synthesis of oligonucleotide fragments, PCR amplification products, can also be used from the genome of the DNA fragments; can be double-stranded, can also be used in the single-stranded DNA or RNA fragments, and the technology to achieve is not subject to the control of patents, and thus the current international development is very fast.
Appliance
Gene chip technology is commonly used in DNA sequencing, gene expression analysis, genome research, genetic diagnosis, drug research and development, as well as industrial and agricultural, food and environmental monitoring and other fields.
Operation method
Gene chip technology
Principle
Gene chips can be categorized into two main types according to their preparation: synthetic gene chips and DNA microarrays. The basic principle of synthetic gene chip is to synthesize oligonucleotides in situ in specific parts of the chip by using techniques such as photolithography. The integration degree of this kind of chip is higher, but the length of the synthesized oligonucleotide probe is shorter, generally 8 to 20 bases, and the longest length does not exceed 50 bases. Therefore, for genes of average length, it is necessary to use multiple probes that overlap each other for accurate identification of the gene. Although the physical integration is high, the integration of biogenetic information is relatively compromised. Moreover, the preparation of such chips is subject to strict patent control, and development is limited.The basic principle of DNA microcollector array is to use a set of special chip-printing device, through the back-and-forth movement of a robotic arm, to curing the pre-prepared gene probes on the surface of the support in an orderly manner by microprinting. Although the integration degree of the chip is relatively low, but the use of the probe group source is more flexible, but the synthesis of oligonucleotide fragments, PCR amplification products, can also be used from the genome of the DNA fragments; can be a double-stranded, can also be used in a single-stranded DNA or RNA fragments, and the technology to achieve is not subject to the control of patents, and thus the current international development is very fast.
Materials and Instruments
Equipment:
①Gene chip printer
②Gene Chip Scanner
③ ultraviolet crosslinker
④ high speed ionizer, constant temperature water bath, high speed vacuum dryer
⑤ Aminosilane-coated slides, slide slots, coverslips
⑥ 384-well plate, 1.5 ml Eppendorf tube (sterilized, those involving RNA need to be treated with 0.1% DEPC)
Reagents:
①Materials: probes, total RNA of cells
② 5xRT buffer, pre-hybridization solution, 2x hybridization solution, isopropanol, anhydrous ethanol, 70%
ethanol, DEPC-H
2
O, sterilized H
2
O
③RNAase inhibitor
④ oligo(dT)18
⑤DTT
⑥ dATP, dGTP, dTTP, dCTP
⑦DMSO
⑧Tris-HCl (pH 6.5)
⑨SuperscrptII reverse transcriptase
⑩Cy3-dCTP, Cy5-dCTP
⑪20 mmol／L EDTA,500 mmol/L NaOH,500 mmol/L HCI,3 mol／L NaAc
⑫ Human Cotl DNA
⑬ Wash A, Wash B, Wash C
Move
The basic process of gene microarray can be divided into the following steps (taking the example of hybridization with expression profiling microarray after labeling sample mRNA by reverse transcription method):
(i) Reagent preparation
(1) Tris-HCl (pH 6.5): Weigh 242.2 g of Tris, add 1600 ml of double-distilled water, heat and stir to dissolve, and prepare Tris mother liquor. Take 80 ml of Tris mother liquor, adjust the pH to 6.5 with hydrochloric acid, add double-distilled water to 100 ml, and sterilize at 103.4 kPa for 20 min.
(2) 5xRT buffer: 250 mmol/L Tris-HCl (pH 8.3), 375 mmol/L KC1, 50 mmol/L MgCl2.
(3) Pre-hybridization solution 25% formamide, 5xSSC, 0.1% SDS, 1% BSA.
(4) 2x hybridization solution 50% formamide, 10xSSC, 0.2% SDS.
(5) Wash A 2xSSC, 0.1% SDS.
(6) Wash B 0.1xSSC, 0.1% SDS.
(7) Wash C 0.1xSSC.
(ii) Preparation of DNA microcollection arrays
(1) probe preparation using conventional molecular biology methods (PCR amplification and gene cloning technology) to prepare the probe, RT-PCR amplified cDNA. after purification, the concentration of 1 μg / μl.
In addition, a positive control probe, a negative control probe and a blank control probe should be prepared. Add DMSO and Tris-HCl (pH 6.5) to the probes at a final concentration of DM-SO (50%), Tris-HC1 (20 mmol/L), and 300 ng/μl of probe, respectively.
The mixture was homogenized and transferred to a 384-well plate. Cover the plate, heat at 95 ℃ for 5 min and place on ice for printing.
(2) Probe printing According to the number of probes, the number of points to be printed for each probe, design the array distribution of probes on the chip, and prepare the corresponding program, and automatically complete the chip printing by the chip printer.
(3) Post-printing processing
① UV cross-linking Place the chip in the UV cross-linking instrument and irradiate it with an energy of 90 mj, so that the DNA is cross-linked on the surface of the slide.
② Fixation Place the chip in the slide box and dry bake at 80 ℃ for 2 h.
(iii) Dry and store away from light.
(iii) Sample preparation
(1) Extract total RNA from tissue cells.
(2) Fluorescent labeling of samples.
① Add 10 μg of total RNA, 2 μg of oligo(dT)18 and DEPC-H2O into a centrifuge tube to a total volume of 14 μl. Heat the above mixture at 70 ℃ for 10 min, and then quickly cool it on ice for 1 min to anneal the oligo(dT)18 and mRNA.
The following components were added: 5xRT buffer, 10 μl; DTT (5 mmol/L), 5.0 μl; RNAase inhibitor (20 U/μl), 1.5 μl; dATP, dGTP, dTTP (25 mmol/L each), 1.0 μl; dCTP (1 mmol/L), 5.0 μl; Cy3/Cy5-dCTP (1 mmol/L, Cy3-DCTP), 5.0 μl. mmol/L, Cy3/Cy5 labeled control and test, respectively), 2.0 μl; Supercrpt II reverse transcriptase (200 U/μl), 1.5 μl; the total volume to 50 μl.
③ Mix well and incubate at 42 ℃ for 2 h to allow reverse transcription of polyA-ized mRNA.
④Shortly centrifuge, and add 1.5 μl of 20 mmol/L EDTA to terminate the reaction.
⑤ Add 2.5 μl of 500 mmol/L NaOH and heat at 70 ℃ for 10 min to degrade the RNA.
(6) Add 2.5 μl of 500 mmol/L HCI to neutralize NaOH.
(7) Add 5 μl of 3 mol/L NaAc and 75 μl of anhydrous ethanol.
⑧ Place at 20 ℃ for 20 min and centrifuge at high speed for 15 min to precipitate cDNA.
⑨ Discard the supernatant and wash the precipitate with 0.5 ml 70% ethanol.
⑩ Dry the precipitate in a high-speed vacuum machine.
Add 10 μl of sterilized deionized water to dissolve and determine the OD260 of the sample.
(D) Molecular hybridization and post-hybridization cleaning
(1) Pre-hybridization
①Carefully take out the chip from the slide box, put it into the prehybridization solution, and incubate it at 42 ℃ for 1 h.
② Pass in sterilized water for 5 times.
③Immerse in isopropyl alcohol for 1 min and air dry.
(2) Hybridization
① Coverslip treatment Rinse with sterilized water, then rinse with anhydrous ethanol, silanize and air dry.
② Sample treatment Take an appropriate amount of each of the purified Cy3 and Cy5 labeled probes and mix them, so that the final concentration of the samples are both 100 ng/μl, then add 1 μl of Cotl DNA (20 μg/μl) and mix gently, then heat at 95 ℃ for 5 min and centrifuge at high speed for 2 min.
③ Mix the probe with an equal volume of 2x hybridization buffer preheated at 42 ℃, and take 4 μl of the treated sample to spot on the array.
④ Carefully cover the coverslip (to avoid air bubbles).
⑤ Add 10 μldH2O into each of the small wells at both ends of the hybridization cassette to maintain the hybridization humidity.
(⑥) Load the chip into the hybridization box, put it into a 42 ℃ water bath, and hybridize for 16～20 h.
(3) Cleaning after hybridization
①Remove the chip from the hybridization box (do not remove the coverslip).
② Immerse the chip into the wash solution A, the coverslip should be made to leave the slide quickly, and gently shake at 42 ℃ for 5 min.
③Transfer the chip to Wash B and shake gently for 10 min at room temperature.
④Remove the chip from Wash B and immediately transfer it to Wash C. Elute for 1 min at room temperature.⑤ Repeat step ④ 4 times.
⑥ Wash in sterilized water for no more than 10 s.
⑦Immersed in anhydrous ethanol.
(viii) Air dry and prepare for scanning.
(E) Scanning of the chip
(1) Scan the chip with the ScanArray Lite Gene Chip Scanner from Gsi Lumonics, and analyze the intensity and ratio of the two fluorescent signals, Cy3 and Cy5, by Quan-tArray software.
(2) The criteria for determining differentially expressed genes included the following two points:① The absolute value of the natural logarithm of the ratio of Cy3 and Cy5 signals is >2.0 (the difference in gene expression is more than 2-fold).
② One of the Cy3 and Cy5 signal values must be 700 or more.
(3) When conducting large-scale gene expression analysis, the data should also be analyzed statistically, such as using cluster analysis, through the establishment of different mathematical models, the results of the abstract data into an intuitive tree diagram, to determine the correlation between the expression of different genes, so as to find information on the function of unknown genes or known genes, or to discover new genes. or discover new genes, etc.
(VI) Experimental results and analysis
The two-color fluorescence scanning images show the results of hybridization of Cy5 and Cy3 labeled probes with the chip, and the color shades indicate the strength of the hybridization signals.
The Cy3 and Cy5 scanning images were completely overlapped and assigned pseudo-colors (Cy5 image in green, Cy3 image in red) to become the result graph of two-probe hybridization, where green indicates high expression, red indicates low expression, and yellow indicates no change in the expression level.
Caveat
(1) DNA probes usually need to be purified before printing to remove enzymes, ions, dNTP, and other substances. The purified product is dried and redissolved in high-salt solution or other denaturing buffer such as 3xSSC.Better hybridization results can be achieved by dissolving the amplified probe in 50% Dimethyl Sulfoxide (DMSO) without using SSC to dissolve it.This is because DMSO denatures the DNA probe and binds better to the slide, providing more single strands to hybridize with the target DNA; and DMSO is hygroscopic and has a lower vapor pressure, allowing the DNA probe solution to be stored longer without significant evaporation during printing.(2) The control probe contains positive and negative control gene fragments and a blank control.The purpose of setting up a positive control is to allow the target genes of different samples to hybridize with this control to obtain essentially the same signal, that is to say, the hybridization signal of the positive control can be used as an internal control for correction (i.e., standardization) during the detection and analysis.The negative control should be selected to avoid homology with the gene under study so as not to produce hybridization signals, but the properties of the nucleic acids [such as length, (G + C) content, etc.] are required to be basically similar; the blank control does not contain any genes, but only the liquid in which these probes are dissolved (i.e., spotting solution) as described above.(3) Before printing, the slides need to be chemically treated on the surface, so that the surface of the slide is derived from amino groups, aldehyde groups, isothiocyanate groups and other reactive groups.These active groups can form ionic bond or covalent bond with the phosphate group, amino group, hydroxyl group and other groups in the DNA molecule, so that the DNA printed on it is firmly cured on the surface of the support to prevent it from being eluted during hybridization.Moreover, the surface of the slide is treated to reduce the diffusion of hydrophilic probes on its surface, thus increasing the print density of the dots. The slide is also treated after printing to immobilize the DNA on the glass surface; it is also necessary to close the unprinted area on the slide to prevent non-specific immobilization of the sample DNA.(4) Pre-printing is also required before formal printing to eliminate differences in the amount of probes initially spotted that are not equal to each other.(5) After labeling, the sample usually needs to be purified before hybridization, otherwise the high fluorescence background during detection will adversely affect the detection of hybridization signals.In order to ensure the sensitivity of microarray detection, special attention should be paid to the extraction, purification and reverse transcription labeling of sample RNA, whose quality and efficiency determine the actual sensitivity of microarray detection.(6) The hybridization process of microarray is basically similar to that of conventional molecular hybridization, and pre-hybridization is carried out first.Pre-hybridization can be closed or inactivated by the free amino group or aldehyde group and other active groups on the slide, otherwise the labeled target DNA of the sample will be non-specifically bound to other sites outside the probe on the slide, thus depleting the target DNA and generating a strong background.In addition unbound DNA probes can be washed away.(7) Factors affecting microarray hybridization include the temperature of the hybridization, the sequence composition of the probe, the concentration of the probe, the concentration of the target gene sequence, and the components of the hybridization solution.Gene expression profiling chip hybridization requires a longer time (often requiring overnight hybridization), high salt concentration, high sample concentration, lower hybridization temperature, but the rigor requirements are lower, which is conducive to improving the specificity of the test, to ensure higher sensitivity and can detect low-copy genes.After hybridization, the chip is washed under rigorous conditions to remove all residues that have not been hybridized. Both hybridization and elution conditions must be optimized experimentally to ensure specificity.(8) When performing the washing of the chip, the coverslip starts to slip If the chip is exposed to air, it is prone to produce high background fluorescence signals; if the coverslip does not slip within 30 s, use tweezers to gently remove it from the surface of the chip; failure to remove the coverslip may reduce the efficiency of hybridization.(9) The fluorescence intensity of the positive hybridization sites on the microarray must be processed and analyzed to identify differentially expressed genes during gene expression profiling. The first step is to normalize the relative fluorescence intensity of each scanning band.Normalization is the correction of differences between the labeling and detection efficiencies of fluorescent markers. These differences can lead to fluctuations in the average Cy5 to Cy3 ratio. It is therefore necessary to correct the fluorescence intensities prior to analysis.
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