DNA MICROARRAY

With the discovery of whole genome constitution of the cells, there is a urgent need to understand how this genes will interact with each other. One method to do this to take “snapshots” of cells, which enables us to measure thousands of genes simultaneously .this process is know as DNA Micrarray/DNA chips/gene chips. Hence microarrays provide a tool for answering a wide range of questions about the dynamics of cells.

Concept of microarray:

The concept of microarray has been originated from southern blot technique. In this a series of high density DNA spots bound to some solid support (usually glass) using automated processes, which allows for much higher and more consistent spot densities to be realized thus increasing the number of samples that can be probed at once . This immobilized DNA molecule can be probed with labeled complementary sequence of DNA. DNA microarrays have spots that contain known sequences of DNA to which labeled sample of unknown composition is hybrized and then detected. Microarray technology is hence based on complementary hybridization mRNA is converted to cDNA using reverse transcription. Types of DNA microarrays: 1. Spotted arrays (cDNA array): They use cDNA probes immobilized to a surface like glass using robot spotting and exposed to a set of targets. The cDNA is put into a PCR plate and loaded onto the robot, this robot deposit the cDNA solution on to glass slides in precise predefined location.

Synopsis Concept of Microarray Types of microarray DNA Microarray Experiment Various methods used in analysis Conclusion

                         Comparing the gene expression between two tissue samples is done using cDNA from each sample and utilizing a separate florescent dye. The two samples are then mixed together and put on the microarrays, each spot on the microarray will hybrids the corresponding cDNA from each sample. If one sample contain more mRNA of a certain gene than the other sample, then the microarray spot for that gene will emit a higher florescent signal .when we sell it under appropriate light source using a high resolution laser scanner and software, the intensity in each channel for each spots are analyzed revealing the difference in expression levels for that gene between two samples.

2. Oligonucleotide arrays:

 They are formed of an array of oligonucleotide(20-80 mu oligos)or peptide nucleic acid probes synthesized either on chip or by conventional synthesis followed by on chip immobilization. Then that array is exposed to labeled sample of DNA and hybridized. By this method we can determine the abundance of complementary sequence.

DNA MICROARRAY EXPERIMENT:  A microarray experiment involves the manufacturing of microarrays and then analyzing hybridization results. The analysis is the more fundamental aspect and is essentially the same regardless of the way they were fabricated.  This microarray experiment will have a glass slide to which single stranded DNA molecules are attached at fixed locations ,to minimize the risk of errors it is essential to keep bacterial culture in an array format, corresponding to the intended positions of the gene on the array ,this experiment typically attempt to compare gene expression levels in different tissues at different times after treatment .

  RNA from the target and control population samples is extracted and labeled with two separate fluorescent labels(Cy3 orCy5) by incorporating the label into copies of the mRNA molecule in the original RNA population ,a single stranded labeled Cdna will be produced complementary to mRNA, the more cdna will be present. Both labeled cDNAs are washed over the microarray.

cDNA sequence corresponding to the extracted RNA hybridize to their complementary sequence corresponding to representative genes. Incorporation of label into each probe is quantified, and probes are diluted so that all are at an equal concentration. Usually a duplicate filter or microarray is prepared for each probe to be assayed. Probes are hybridized separately with each array. Filters arrays are incubated with probe and washed for glass microarray, hybrization is done under a cover slip and subsequently dipping them into wash solutions. the dyes enable the amount of sample bound to a spot to be measured from the level of fluorescence emitted when a laser excites it ,hydridized probe is detected by either chemiluminescence’s or direct UV fluorescence for microarray,thus the relative expression levels of the genes in the sample and in control population can be estimated from the fluorescence intensities and colors for each spot based on the amount of probe hybridized to each target spot, information is gained about the specific mRNA composition and representative in the sample. The major advantage of gene arrays is that they can provide information on thousands of targets in a single experiment and huge amount of data can be gained.

Analysis of data:

 Several computational tasks can be performed with the data and then used for various applications like clustering genes, for identifying which genes seem to be regulated together. Clustering samples for identifying which treatments/individuals have similar profiles. For classifying genes to know their function etc. 

Various methods used in analysis of data:

1. Image analysis: to analyse data images produced by processing from laser scanner to decide the fluorescence intensity for each spot.aligning the image with a grid representing the array layout identifies each spot and their boundaries are decided and then the intensity for the red and green channels arises.

2. Normalization: this is the process of correction for systematic errors,while they can be random errors that cannot be corrected .normalization is performed during scanning ,when the power of the laser is adjusted so that the intensities of red and green channels fall in the same range, there are three ways to normalize gene expression data from single array hybrization.

1. Total intensity normalization

2. Normalization using regression techniques

3. Normalization using ratio statistics 

3. Clustering the gene expression profiles:

Clustering is an exploratory data analysis tool that can help to understand the general characteristics of data and to help visualize the data and identify the patterns in gene expression data.

4. Principal component analysis (PCA):

PCA is an exploratory technique and is used to visually estimate the number of clusters represented in the data.PCA is a powerful technique for the analysis of gene-expression data when used with another classification technique, such as K-means clustering or SOMs that requires the user to specify the number of clusters this can be used to specify k and to group genes into related clusters. It is a mathematical technique that picks out relevant patterns in the data and reduces the effective dimensions of gene expression space without significant loss in information.

Conclusion:

So the above explained process of microarray technique plays a vial role in analyzing the genes at molecular level at a faster pace and in huge amounts.infact this gene arrays provide information on thousands of targets in a single experiment, thus DNA microarray is a revolutionary new method in the field of molecular biology.

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