文档介绍：MethodsMethods inin MolecularMolecular BiologyBiologyTM
VOLUME 224
FunctionalFunctional
GenomicsGenomics
MethodsMethods andand ProtocolsProtocols
EditedEdited byby
MichaelMichael . BrownsteinBrownstein
ArkadyArkady . KhodurskyKhodursky
Fabrication of cDNA Microarrays 1
1
Fabrication of cDNA Microarrays
Charlie C. Xiang and Michael J. Brownstein
1. Introduction
DNA microarray technology has been used essfully to detect the
expression of many thousands of genes, to detect DNA polymorphisms, and
to map genomic DNA clones (1–4). It permits quantitative analysis of RNAs
transcribed from both known and unknown genes and allows one pare
gene expression patterns in normal and pathological cells and tissues (5,6).
DNA microarrays are created using a robot to spot cDNA or oligonucleotide
samples on a solid substrate, usually a glass microscope slide, at high densities.
The sizes of spots printed in different laboratories range from 75 to 150 µm
in diameter. The spacing between spots on an array is usually 100–200 µm.
Microarrays with as many as 50,000 spots can be easily fabricated on standard
25 mm × 75 mm glass microscope slides.
Two types of spotted DNA microarrays are mon use: cDNA and
synthetic oligonucleotide arrays (7,8). The surface onto which the DNA is
spotted is critically important. The ideal surface immobilizes the target DNAs,
and patible with stringent probe hybridization and wash conditions (9).
Glass has many advantages as such a support. DNA can be covalently attached
to treated glass surfaces, and glass is durable enough to tolerate exposure
to elevated temperatures and high-ionic-strength solutions. In addition, it is
nonporous, so hybridization volumes can be kept to a minimum, enhancing the
ics of annealing probes to targets. Finally, glass allows probes labeled with
two or more fl uors to be used, unlike nylon membranes, which are typically
probed with one radiolabeled probe at a time.