November 2014 – Bio-Resource

DNA microarray is a hybridization technique performed in microscopic glass slide with surface modification, anchoring specific set of probes sequences complementary to the Target. Upon complementation with the target sequence fluorescence signal can be captured and state of the test sample can be identified. DNA microarrays can be used to measure changes in expression levels, to detect single nucleotide polymorphisms (SNPs), or to genotype or targeted resequencing. Principle of DNA Microarray The principle behind microarray is the hybridization between two DNA strands. Complementary sequence of the DNA pair specifically by forming hydrogen bonds, the more number of complementary base pairs in a nucleotide sequence will have a tighter non-covalent bonding between the two strands. After the hybridization of target DNA to the probes attached on the glass slide, slides are washed to remove any non-specifically bound sequences, only strongly paired strands will remain hybridized. Fluorescently labelled target sequences that bind to a probe sequence generate a signal that depends on the hybridization conditions (such as temperature), and washing after hybridization. Total strength of the signal, from a spot (feature), depends upon the amount of target sample binding to the probes present on that spot. Microarrays use relative quantitation in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position. Preparing Glass Slide for Surface Modification Glass slide used for surface modification microarray need to be clean, to remove any organic contaminats and dirt glass can be cleaned with Ethanol, Toulene or Etching with pirhana. Etching with pirhana (7:3 H2SO4:H2O2) is the better choice among the other cleaning methods. One need to be careful while while handling pirhana solution as it is a strong oxidizer. Surface Modification Strategies Various Surface modification are developed for DNA microarray, few of the most commonly used ones are Poly – L- Lysine, 3-aminopropyltrimethoxysilane (APS), 3-glycidoxypropyltrimethoxysilane (GPS) and Aldehyde or carboxylic acid. Oligo attachment to the modified glass surface Oligo attachment to PLL coated glass slide Poly – L – Lysine hydrogen bonding with an oligonucleotide. Oligo attachment to APS coated glass slide 3-aminopropyltrimethoxysilane (APS) hydrogen bonding with an oligonucleotide. Oligo attachment to GPS coated glass slide 3-glycidoxypropyltrimethoxysilane (GPS) covalently bound to an amine-terminated oligonucleotide. Oligo attachemnt to DAB Dendrimer modified glass slide One-half of a DAB dendrimer hydrogen bonding with an oligonucleotide Spotting Oligo Probes on the glass slide Mechanical Spotting Ink Jetting Microarray Workflow Microarray Detection / Capture List of commercially availbale DNA Microarrays ACLARA Bio Sciences GeneChipTM - Affymetrix LabChip - Caliper Technologies LabCD system - Gamera Bioscience Genetix Ltd Agilent Illumina Incyte Microarray Systems Nanogen Sequenom and GenoSensor - Vysis Inc. (Downers Grove, IL) References Technical Resources, Array It Corp Technical Resources, Affymetrix A Beginer's Guide to DNA microarrays Got something to say about this post? Leave a comment...your comments are valuable for improving the posts.

Month: November 2014

DNA Microarray – Types, Strategies and applications

Comparison of TAE and TBE Buffers used in Gel Electrophoreis – Advantages & Disadvantages

DNA Topology – Enzymes and Modes of action on cccDNA