Gene Cloning

The Analysis of the Regulation of Gene Expression 355
are often termed DNA micro-arrays (nylon based arrays are often called
macro-arrays). DNA micro-arrays are printed by specialized machines that
deposit a single spot of each probe at a mapped position on the glass slide
so that, following hybridization, you know which spot corresponds to each
gene.
The DNA probes bound to the slide can be either oligonucleotides or
PCR fragments. PCR fragments can either contain the whole coding
sequence (ORF) or a shorter region within the gene. The PCR products
need to be denatured to give single-stranded DNA when spotted onto the
glass slide or nylon membrane in order to allow hybridization of the
labeled cDNA. One disadvantage of using the complete ORF is that it may
lead to cross-hybridization between cDNAs and probes for homologous
genes leading to incorrect readout for the homologous genes. PCR primers
could be designed that direct the amplification of a unique region for each
gene, however this would require a large amount of bioinformatic analysis.
Whether you amplify the whole ORF or a section of the gene, probe preparation
requires a large number of PCR reactions; for example, a yeast array
would require more than 6000 reactions. This has led to automation of
many procedures with laboratory robots being used to set up reactions. In
addition, if gene specific primers are being used you need to synthesize a
large number of oligonucleotides; 12,000 are required to amplify the 6000
yeast genes. In many cases the DNA probes are amplified from an existing
library of clones. If this is the case it may be possible to use the same
primers, derived from the vector sequence, to amplify all the probes. For
example, a library containing every E. coli gene cloned into the same vector
has been constructed and so it is possible to use primers that anneal to
the vector sequence on either side of the insert. However, you still need to
set up thousands of PCR reactions and, as the library was originally created
by using gene specific primer pairs to amplify each ORF, if starting from
scratch it would not save time or money. The alternative is to use oligonucleotide
probes. Oligonucleotide probes are normally 40 to 70 bases in
length and are designed so that they are unique for each gene to avoid
cross-hybridization with homologous genes. Gene chips are another type
of array. These are created by synthesizing oligonucleotides de novo
directly on the array using photolithographic technology, the same technology
that is used to make electronic chips. The oligonucleotides are
approximately 25 bases in length and normally several oligonucleotides are
synthesized for each gene.
Once an array has been constructed, the basic experimental procedure
for doing a transcriptomic analysis is the same whether you are using a
nylon-, glass- or gene chip-based array. RNA is isolated from test and control
cells. Labeled cDNA is then synthesized from this RNA and hybridized
to the array, and the amount of cDNA bound to each spot is quantified. If
using a nylon-based array the cDNA can be labeled with a radioisotope and