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Microarray Technique
An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules. An array experiment makes use of common assay systems such as microplates or standard blotting membranes. The sample spot sizes are typically less than 200 microns in diameter usually contain thousands of spots. Thousands of spotted samples known as probes (with known identity) are immobilized on a solid support (a microscope glass slides or silicon chips or nylon membrane). The spots can be DNA, cDNA, or oligonucleotides. These are used to determine complementary binding of the unknown sequences thus allowing parallel analysis for gene expression and gene discovery. An experiment with a single DNA chip can provide information on thousands of genes simultaneously. An orderly arrangement of the probes on the support is important as the location of each spot on the array is used for the identification of a gene.
Types of Microarrays
Depending upon the kind of immobilized sample used construct arrays and the information fetched, the Microarray experiments can be categorized in three ways: 1. Microarray expression analysis: In this experimental setup, the cDNA derived from the mRNA of known genes is immobilized. The sample has genes from both the normal as well as the diseased tissues. Spots with more more intensity are obtained for diseased tissue gene if the gene is over expressed in the diseased condition. This expression pattern is then compared to the expression pattern of a gene responsible for a disease. 2. Microarray for mutation analysis: For this analysis, the researchers use gDNA. The genes might differ from each other by as less as a single nucleotide base. A single base difference between two sequences is known as Single Nucleotide Polymorphism (SNP) and detecting them is known as SNP detection. 3. Comparative Genomic Hybridization: It is used for the identification in the increase or decrease of the important chromosomal fragments harboring genes involved in a disease.
Applications of Microarrays
Gene discovery: DNA Microarray technology helps in the identification of new genes, know about their functioning and expression levels under different conditions. Disease diagnosis: DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in
the tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer. Drug discovery: Microarray technology has extensive application in Pharmacogenomics.Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect. Toxicological research: Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants.
GEO
In the recent past, microarray technology has been extensively used by the scientific community. Consequently, over the years, there has been a lot of generation of data related to gene expression. This data is scattered and is not easily available for public use. For easing the accessibility to this data, theNational Center for Biotechnology Information (NCBI) has formulated the Gene Expression Omnibusor GEO. It is a data repository facility which includes data on gene expression from varied sources.
For 35-45 mers Parameter Probe Length Probe Length tolerance Probe Target Tm Minimum Value 10 0 40 Maximum Value 99 15 99 Default Value 40 3 70 C Unit bases
99 99.9 99.9 99
For 65-75 mers Parameter Probe Length Probe Length tolerance Probe Target Tm Probe Tm Tolerance (+/- above) Hairpin Max G Self Dimer G Run/Repeat Minimum Value 10 0 40 0.1 0.1 0.1 2 Maximum Value 99 15 99 99 99.9 99.9 99 Default Value 70 3 75 5 6 8 6 Kcal/mol Kcal/mol bases C Unit bases
Other Parameters
Probe Location 1. 3' end bias: The oligos chosen should be towards the 3' end of the gene i.e. Default : 3' end. 2. The oligos should be designed by default within 999 bases of 3' end. The range can be from 0 to 1500 bases. The oligos should be free of cross homology (i.e They should be BLAST searched against the appropriate genome category). DNA Microarray with Array Designer: Array Designer is an exceptional software to design highly specific oligos for expression and SNP genotyping microarray experiments.
For cross species assays, AlleleID identifies the conserved regions to design a universal probe. For related organisms, AlleleID can be used to study gene expression when genome draft of the organism under study is not available. This powerful functionality is sure be helpful for many challenging tasks such as detection, identification, quantification or monitoring of contaminants, environments...
Extensive support for real time or quantitative PCR Assays & SNP/expression microarrays
AlleleID designs optimal SYBR Green primers, TaqMan probes, TaqMan Minor Groove Binding (MGB) probes, FRET probes or molecular beacons for real time quantitative PCR differential gene expression and SNP genotyping assays. You can also design real time PCR primers and dual labeled probes for up to ten thousand sequences in a single run for making SNP detection or expression microarrays.
Multiplex ligation-dependent probe amplification (MLPA) assays for copy number and mutation detection
AlleleID is the only program that designs synthetic probes for MLPA or Multiplex Ligation-dependent Probe Amplification technique introduced by MRC Holland enabling the researcher to extend the technique when appropriate kits are unavailable. It is a rapidly growing high throughput method for genetic profiling. It can detect exon deletions, copy number changes, and CpG methylation patterns. It is cost-effective, sensitive, specific and reproducible. AlleleID also designs mRNA specific, DNA specific and mutation specific MLPA probes for PamGene Arrays. This will enable PamChip users designing their own probes to detect genes of interest using PamGene's high precision detection platform. Combining these two technologies, copy number changes and mutation can be easily detected in just 6 hours. Please browse through the feature list of AlleleID to learn more about what the program can do for you. To give you a quick start, we have prepared a multimedia tutorial. It steps you through each function of the program.
AlleleID Features -Real Time PCR Primer Design -SYBR Green Primer Design
AlleleID Highlights -Cross Species/ Taxa Specific Assays -Species Identification Assays
-TaqMan Probe Design -Molecular Beacon Design -FRET probe design -Microarray Probe Design -Database Support -Input & Output Formats -Microarray Based Gene Splicing Detection -Exon Junction Primer Design -MLPA Probe Design -Luminex xMAP Assays
Alternative Splicing
DNA microarray is perhaps the most useful technology for splice variant studies. AlleleID includes the ability to design microarray probes for detecting alternative splicing and gene splicing events.
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AlleleID designs two types of microarray probes that we call junction probes and exon probes, giving the user complete control over the design parameters. AlleleID detects the alternative splicing events by designing probes for each junction and/or for each splice variant created. AlleleID designs exon probes. Such probes are important to confirm the presence of an exon in the variant. Technical resource on Gene Splicing.