Wednesday, June 13, 2007

DNA Microarray

DNA Microarray - A technology that is reshaping molecular biology

It is widely believed that thousands of genes and their products (i.e., RNA and proteins) in a given living organism function in a complicated and orchestrated way that creates the mystery of life. However, traditional methods in molecular biology generally work on a "one gene in one experiment" basis, which means that the throughput is very limited and the "whole picture" of gene function is hard to obtain. In the past several years, a new technology, called DNA microarray, has attracted tremendous interests among biologists. This technology promises to monitor the whole genome on a single chip so that researchers can have a better picture of the interactions among thousands of genes simultaneously.

Terminologies that have been used in the literature to describe this technology include, but not limited to: biochip, DNA chip, DNA microarray, and gene array. which refers to its high density, oligonucleotide-based DNA arrays. However, in some articles appeared in professional journals, popular magazines, and the WWW the term "gene chip(s)" has been used as a general terminology that refers to the microarray technology. Affymetrix strongly opposes such usage of the term "gene chip(s)". More recently, I prefer the term "genome chip", indicating that this technology is meant to monitor the whole genome on a single chip. GenomeChip would also include the increasingly important and feasible protein chip technology.

Base-pairing (i.e., A-T and G-C for DNA; A-U and G-C for RNA) or hybridization is the underlining principle of DNA microarray.

An array is an orderly arrangement of samples. It provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identifying the unknowns. An array experiment can make use of common assay systems such as microplates or standard blotting membranes, and can be created by hand or make use of robotics to deposit the sample. In general, arrays are described as macroarrays or microarrays, the difference being the size of the sample spots. Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contains thousands of spots. Microarrays require specialized robotics and imaging equipment that generally are not commercially available as a complete system.

DNA microarray, or DNA chips are fabricated by high-speed robotics, generally on glass but sometimes on nylon substrates, for which probes* with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously - a dramatic increase in throughput. There are two major application forms for the DNA microarray technology:

1) Identification of sequence (gene / gene mutation) and

2) Determination of expression level (abundance) of genes.

There are two variants* of the DNA microarray technology, in terms of the property of arrayed DNA sequence with known identity:

Format I: probe cDNA (500~5,000 bases long) is immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method, "traditionally" called DNA microarray, is widely considered as developed at Stanford University. A recent article by R. Ekins and F.W. Chu seems to provide some generally forgotten facts.

Format II: an array of oligonucleotide (20~80-mer oligos) or peptide nucleic acid (PNA) probes is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences are determined.

The microarray (DNA chip) technology is having a significant impact on genomics study. Many fields, including drug discovery and toxicological research, will certainly benefit from the use of DNA micro array technology.

Design of a DNA Microarray System

There are several steps in the design and implementation of a DNA microarray experiment. Many strategies have been investigated at each of these steps. 1) DNA types; 2) Chip fabrication; 3) Sample preparation; 4) Assay; 5) Readout; and 6) Software (informatics)

Table 1. Steps in the design and implementation of a DNA microarray experiment

Probe (cDNA/oligo with known identity)

Chip fabrication
(Putting probes on the chip

Target (fluorescently labeled sample) Assay Readout Informatics
Small oligos,
(whole organism on a chip?)

Photolith-ography, pipette, drop-touch, piezoelectric (ink-jet), electric, ...

RNA, (mRNA==>)

Hybridization, long, short, ligase, base addition, electric, MS, electrophoresis, fluocytometry, PCR-DIRECT, TaqMan, ...

Fluorescence, probeless (conductance, MS, electrophoresis), electronic, ...

Robotics control,

Image processing, DBMS, WWW, bioinformatics, data mining and visualization

There are so many options and combinations,as can been seen from the number of companies involved in this business. It seems too early to judge who will be the winner(s) in this game. The forecast is further complicated by recent fights among companies on intellectual property issues.

Applications of DNA Microarray Technology

Disease diagnosis

Many "microfluidics" devices fall in this category. Although they are not the "traditional" gene chip or microarray, I decided to list related links at this site because of their close connection and integration to the gene chip (microarray) technology.

Drug discovery: Pharmacogenomics

Why some drugs work better in some patients than in others? And why some drugs may even be highly toxic to certain patients? My favorite definition (modified): Pharmacogenomics is the hybridization of functional genomics and molecular pharmacology. The goal of pharmacogenomics is to find correlations between therapeutic responses to drugs and the genetic profiles of patients.

Toxicological research: Toxicogenomics

Have you seen anybody using this terminology? Now let's try to give it a definition: Toxicogenomics is the hybridization of functional genomics and molecular toxicology. The goal of toxicogenomics is to find correlations between toxic responses to toxicants and changes in the genetic profiles of the objects exposed to such toxicants.

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