DNA MICROARRAY OR GENE CHIP OR BIO CHIP
11. DNA MICROARRAY OR GENE CHIP OR BIO CHIP
DNA microarray technology is used for analysis of thousands of gene’s expression within parallel fashion. It is used for detecting known and unknown function, detecting polymorphisms and detecting genomic DNA mutation both in prokaryotic and eukaryotic DNA. DNA microarray is known as a systematic preparation of thousands of recognized gene which are sequenced and printed as spot on a solid support in the rows and columns. Spots are arrayed in an orderly manner. The solid support is impermeable and generally made up of glass (glass slide which is encased in plastic), silicon chips or nylon membrane. Printed gene sequence on spot is complementary to the desired gene need to be identified. The printed sequence of the gene could be an oligonucleotide, which is chemically synthesized or products of PCR like cDNA.
DNA microarrays are two types
11.1. cDNA microarrays
This was first shown by Patrick Brown. Glass cDNA microarray contains a glass slide possessing 10,000-20,000 spots. Each spot represents a specific cDNA sequence having the concentration of 100-500 µg/ml along with the diameter of 50-150 µm and between two spot distance is maintained. These spots are formed by the high-speed precision robot. The area of each spot( square) is 3.6 cm2. For solvents glass slide possess chemical resistance. Glass slide having less intrinsic fluorescence and fine mechanical constancy due to high thermal strain point.
The first step is the selection of material for spot and then purification of the desired gene from that material. The gene is amplified as cDNA from PCR through universal primer or gene-specific primers, purity of cDNA is checked by sequencing and concentration is measured by agarose gel. All amplified product should be of same size, concentration and kinetic properties. Glass slide is modified generally by poly L-lysine (form an ionic bond with DNA) or other cross-linking chemicals such as polyethyleneimine polymer to cause immobilisation of cDNA on the slide through a covalent or non-covalent bond. After modification pre-fabricated cDNA micro spot are placed on the glass slide by robotic pins or inkjet printing. After that DNA is dried at room temperature overnight and binding of DNA is prevented by UV cross-linking and this decreases background signal upon hybridization of the target, which is labelled. In this case low density array form.
Advantages and disadvantages of cDNA microarrays
1. Lower cost specific equipment is not required
2. Increased detection sensitivity because able to detect target sequences up to 2 kbp and data capture by equipment already available in the laboratory and this flexibility of design is demanded by the scientific goals of the experiment.
3. Disadvantage synthesis, purification and storage before microarray fabrication of cDNA require more labour and cross-hybridisation also a major problem.
11.2. In situ oligonucleotide microarray
This was first developed by Stephen Fodor (1991). In situ chemical synthesis of oligonucleotide (short single strands of DNA) array by the use of photolithography. This is also called Gene Chips. The DNA arrays are based on the high-density oligonucleotide. There are many spots on the plate. Each spot contains a unique DNA sequence in single-stranded form.
Affymetrix's GeneChip's fabrication process involves the addition of the nucleotide to form oligonucleotide strand on quartz wafer and involvement of laser light, which help to adhere nucleotide on wafer’s solid surface, thus called photo (laser light) litho (solid surface) graph.
11.3. Fabrication process
There are 10,000-20,000 spots on the chip and each spot contain different DNA sequence like the first spot contain AGACT sequence second spot contain AGACA sequence, the third spot contain ACATA sequence, the fourth spot contain GATAT......... As seen here first, second and third has same nucleotide A as starting nucleotide, dATP is added in first, second and third spot and block the fourth spot by minute mask, after that laser light is provided to first, second and third spots, which cause immobilization of ATP in first, second and third spot box. Then washing's done to first, second and third which cause the removal of excess or non-immobilized dATP.
Now, first, the second and third spot is blocked by minute mask and dGTP is added in fourth spot and laser light is provided to cause immobilization of a GTP. Then washing is done to the fourth spot, which causes the removal of excess or non-immobilized dGTP.
Now from here, each nucleotide added has a modification of 3’OCH3 instead of 3’OH, which is present in unmodified nucleotide and require to form a phosphodiester bond. This nucleotide modification prevents the formation of the phosphodiester bond between added nucleotide because from this step laser is not provided for immobilization rather DNA polymerase is added and used to make the phosphodiester bond between the immobilized first nucleotide and added modified nucleotide. This process is called nucleotide blocking. As phosphodiester bond is formed, washing is carried out to remove non-phosphodiester bond forming a nucleotide, then demethylase is added to remove the methyl group from 3’ end and create 3’OH from 3’OCH3. Now, this nucleotide is able to form the phosphodiester bond with next modified nucleotide added.
In the next step 3 and 4 boxes is blocked by the minute mask and modified GTP (3’OCH3) added to 1 and 2 box and DNA polymerase also added, which form a phosphodiester bond between the immobilized first nucleotide and added modified nucleotide GTP. Washing is carried out to remove non-phosphodiester bond forming GTP (3’OCH3) nucleotide, then demethylase is added to remove the methyl group from 3’ end and create 3’OH from 3’OCH3. Now, this nucleotide GMP 3’OH is able to form a phosphodiester bond when next modified nucleotide added. By continuously repeating this process, an oligonucleotide strand of 25 nucleotide sequence is formed in each square or box.
Speed, reproducibility and specificity are the advantages of this format and affordability and flexibility are the disadvantage of this format.
11.4. Principle and steps of DNA Microarray experiments
The Basic principle of this technology is that the immobilized cDNA or oligonucleotides, also known as reporter component, has a complementary sequence to our desired gene which is present within the sample. This gives information that our desired gene is present within the sample or not.
Four major steps of this process
11.4.1. Construction of microarray, which can be any cDNA or oligonucleotide is the fabrication process.
11.4.2. Sample preparation and fluorescent labelling
In the microarray, we can compare the gene expression pattern between the liver cell and skin cell or mutant cell and normal cell or in a specific condition, which is called query/experimental sample with the expression of all gene present in cells, which is called control. Here we compare the expression profiles of genes in isogenic mutant or bacteria within infected cells (query) and wild type strain or normal bacteria (control). mRNA is isolated from both type of cells through affinity chromatography and mRNA is converted into cDNA with the help of reverse-transcriptase enzyme and then mutant cells cDNA are fluorescently labelled with red fluorescent dyes and wild type cDNA are fluorescently labelled with green fluorescent dyes. Example of fluorescent dyes - cyanine dyes (Cy3 and Cy5), rhodamine.
Incubation of microarray slide within bovine serum albumin (BSA), saline-sodium buffer (SSC) and Sodium Dodecyl Sulfate (SDS) is done at high temperature to prevent the nonspecific binding. After this step cDNA of mutant cells which are fluorescent labelled with red fluorescent dyes and wild type cDNA which are fluorescent labelled with green fluorescent dye are poured on DNA microarray and then hybridization takes place between complementary sequence of fluorescently labelled cDNA of mutant and wild type cell with immobilized cDNA or oligonucleotide array present on slide or wafer. After this step washing is done at stringent condition (high temperature and low ionic strength) with wash buffer to remove non hybridized fluorescent labelled cDNA of mutant and wild type and prevent cross-hybridization.
11.4.4. Analysis of Data and Image acquisition
The final step of this microarray experiments is data analysis and image acquisition. In this step firstly slide is dried and then placed under the laser scanner to identify the amount of hybridized fluorescent labelled cDNA with cDNA spot or oligonucleotide. An emission with characteristic spectra is generated due to laser excitation of the incorporated targets and this is examined by a confocal laser microscope.
Characteristically, microarray software frequently uses three colour spots. Control is compared with Upregulated gene shown by green spot. And with downregulated gene shown by the red spot. The genes which display equal quantity in both experimental and control samples are shown by yellow spot.
11.5. Applications of DNA Microarray Technology
DNA microarray technology is used for the comparison of the profile of gene expression between different cell populations. It is used to identify different type of mutation in the gene, thus act as a comparative genomics tool.
- TOOL AND TECHNOLOGY
- HYBRID PLASMID / PHAGE VECTORS
- ARTIFICIAL CHROMOSOMES
- SHUTTLE VECTORS
- ENZYMES USED FOR RECOMBINANT DNA TECHNOLOGY
- DNA LIBRARY
- FLUROSCENT ACTIVATED CELL SORTER
- DNA MICROARRAY OR GENE CHIP OR BIO CHIP
- ANTIBODY GENERATION
- RADIOIMMUNOASSAY (RIA)
- ELISA OR ENZYME LINKED IMMUNOSORBANT ASSAY
- POLYMERASE CHAIN REACTION
- TYPE OF HYDROLYSIS PROBE
- X-RAY DIFFRACTION
- NMR (NUCLEAR MAGNETIC RESONANCE)
- CIRCULAR DICHROISM
- DNA SEQUENCING
- TRANSGENIC ANIMALS
- CRE–LOX P RECOMBINANT SYSTEM
- GENE THERAPY
- TRANSGENIC PLANTS
- PLANT TISSUE CULTURE (PTC)
- MICRO PROPAGATION
- ARTIFICIAL SEEDS
- PRACTICAL APPLICATIONS OF PLANT TISSUE CULTURE
- ANIMAL CELL CULTURE