Introduction of DNA Microarray
Earlier, genes and their expression profiles have been studied on an individual basis. DNA Microarray technology now allows us to look at many genes at once. It allows a quantitative and qualitative comparison between the gene expression patterns of two cells.
Definition of DNA microarrays
Array means to place in an orderly arrangement and also called DNA chips. Other names of these chips are gene chips and biochips too. DNA fragment representing a gene is assigned a specific location on the array. Location of each spot – use to identify a particular gene sequences. 30000 spots can be placed in one slide.
Principle of DNA microarrays
Principle Based on hybridization probing uses fluorescently labeled nucleic acid molecules- “mobile probes” Spots are single stranded DNA fragments, strongly attached to the slide. RNA or cDNA is tagged with a fluorescent dye.
Probe – a standardized set of DNA sequences. Target or sample – labeled
experimental DNA or RNA. Autoradiography laser scanning fluorescence detection devises Enzyme detection system.
Detection methods Hybridization method in DNA Microarray
Target DNA is labeled and incubated with microarray. For the detection of
hybridization pattern – reverse dot blot is used probe high GC content – hybridized more strongly than those with high AT content.,matching the target will hybridized more strongly than will probes with mismatches, insertions and deletions. Radio active and non-radioactive methods-It involve biotin or digoxigenin labeling require direct detection through, autoradiography gas phase ionization, phase ionization phosphoimagers.
Fluorescence detection method in DNA Microarray
Multiplexing- one target DNA may be labeled with more than one fluorochromes . Hybridization can be screened using automatic scanners.
Characteristic features of DNA chips:
Parallelism– allows parallel acquisition and analysis of massive data and a
meaningful comparison between genes or gene products represented in microarray.
Miniaturization–involves miniaturization of DNA probes and reaction volumes thus reducing time and reagent consumption. Multiplexing –it involve multicolor fluorescence allow comparison of multiple samples in a single DNA chip. Automation–manufacturing technologies permits the mass production of DNA chip and automation leads to proliferation of microarray assays by ensuring their quality, availability and affordability Types of DNA chips Two types-
- Oligonucleotide based
- chips cDNA based chips
Technical application in DNA Microarray
Green represents control DNA, where either DNA or cDNA derived from normal tissue is hybridized to the target DNA. Red represents Sample DNA, where either DNA or cDNA derived from diseased tissue hybridized to the target DNA. Yellow represents a combination of control and sample DNA, where both hybridized equally to the target DNA. Black represents areas where neither the control nor the sample DNA hybridized to the target DNA.
Probes are oligonucleotides ,cDNA or small fragments of PCR products that
correspond to mRNAs and are spotted onto the microarray surface.
There are commercially available designs that cover complete genomes from
companies such as GE Healthcare, Affymetrix ,Ocimum Biosolutions ,or Agilent.
Applications of DNA microarray
- Detection of single nucleotide polymorphisms (SNPs) Characterization of mutant
- Genetic mapping
- In cancer
Tumor formation involves simultaneous changes in hundreds of cells and variations in genes.
Gene microarrays have been used for comparative genomic hybridization. In this technique, genomic DNA is fluorescently labeled and used to determine the presence of gene loss or amplification
The conversion of a non-invasive tumor to an invasive tumor also warrants research.
- Antibiotic treatment
- Early detection of oral precancerous lesions
- PRINCIPLE OF BASIC MOLECULAR BACTERIOLOGY-SHAKIBAIE MR. (Ph.D)
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