Target Discovery and Validation Reviews and Protocols

Gene Target Discovery 121
There is no doubt that a band pattern showing potentially differentially
expressed transcripts may be obtained in a short time. However, the most timeconsuming
and problematic step in the overall DD technique is the confirmation
that the isolated cDNA clone represents a differentially expressed gene.
This problem has led to a multitude of publications reporting improvements and
simplification of the basic protocol to reduce the high frequency of false positives
and to confirm the differential expression pattern of selected candidates
(27–30). A high rate of false positives and time-consuming effort to find the true
positives have been the main drawbacks with this method (31,32). In addition,
rare transcripts are difficult to detect.
The advances are the experimental simplicity by which candidates are
obtained, the ability to use as little as a few nanograms of starting material (33),
and the possibility to identify novel transcripts.
1.2. Global Techniques
Global cDNA techniques analyze the total or large parts of the mRNA populations
in a single assay. The aim may be to quantitatively estimate the number
of expressed genes in a defined biological material, estimate their absolute
or relative abundance levels, survey global changes in gene expression patterns
in response to extrinsic and intrinsic factors, identify specific genes whose
expression is altered because of such factors, or simply to identify as many as
possible of the genes expressed in a defined biological material. This process
may or may not include differentially expressed, novel as well as known genes.
Because of the unstable nature of RNA, the mRNA populations are usually
reverse transcribed into DNA complementary to the RNA strand (cDNA),
which in theory has the same distribution as the original mRNA populations. It
is the populations of cDNA that normally are the subjects of investigation.
1.2.1. Large-Scale Sequencing of Expressed Sequence
Tag (EST) Sequences
ESTs are segments of sequences from cDNA clones that correspond to mRNA.
The tags can thus be used to identify transcripts in a specific tissue. Adams
and co-workers coined the term EST in 1991, when the first high-throughput
sequencing of a cDNA library was reported (34). The clones are randomly picked
and either randomly single-pass sequenced, or more commonly, sequenced from
the 5′ or 3′ ends or both, to generate on average a 200- to 700-bp sequence. Even
much smaller segments are sufficient for rapid identification of expressed genes
by sequence analysis. The initial objective of EST sequencing was to speed
up the process of gene identification and possible splicing alternative events
(35). In addition, large-scale sequencing of EST sequences has proven to be