Bioactive Agents from Natural Sources: Trends in Discovery and Application - Advances in Biochemical Engineering Biotechnology - page 131

5.1.1
Analytical Methods

The revolution in molecular biology gave access to new techniques enabling
the elucidation of disease-causing genes as potential new targets for therapy.
Positional cloning, e.g. relies on the discovery of an association between a
phenotype and a DNA marker in affected families or animals [323]. By sub-
sequent analysis and gene sequencing a candidate gene can be revealed,as it was
shown for the identification ofthe genetic defects underlying the human mono-
genic disorders cystic fibrosis [324] and Huntington's disease [325],or the recent
discovery of the murine gene responsible for obesity [326].
The ultimate objective of the international Human Genome Project schedu-
led for the year 2005 is to determine the DNA sequence of the approximately
3 10 9 base pairs of the haploid human genome. However, reading the DNA

sequence will not result in information on gene functions.Consequently,a great
deal of effort is being expended especially on the localization and functional
analysis of genes potentially related to disease phenotypes [323].
Expressed sequence tags (ESTs) e.g.represent a special class ofDNA markers
[327] derived from spliced mRNA.Such cDNA copies of mRNA can be genera-
ted from cells and tissues by PCR (polymerase chain reaction) techniques.
Transfer into the bacterium Escherichia coli gives access to a cDNA library
consisting of more than 10 6 clones that represent the entire repertoire of genes
expressed in the tissue ofinterest at the time ofisolation.The nucleotide sequen-
ce can be determined for short regions of individual cDNAs,and this provides a
unique identifier, or EST, for that cDNA representing a specific gene.At insti-
tutions such as Human Genome Sciences (HGS), The Institute for Genomic
Research (TIGR), Incyte, and Washington University together with Merck are
applying the EST strategy coupled to powerful computation to robotic high-
throughput technologies towards the determination ofthe nucleotide sequences
of thousands of clones within days.However,an EST by itself frequently cannot
be ascribed a precise function, but a novel DNA sequence can be analyzed by
comparison of its nucleotide sequence within a databank of known sequen-
ces. In case of homology with known superfamilies of genes, such as protein
kinases, cytokines, or G-protein-coupled 7-membrane-spanning receptors, a
related function can be assigned [323].
Alternative techniques such as subtractive hybridization and differential
display cloning [328],however,lead directly to the identification of genes that
underly phenotypes.These techniques focus exclusively on genes that are differ-
entially expressed in different cell or tissue samples.Differential display cloning
permits a simultaneous comparison of genes that are either up-regulated or
down-regulated within different mRNA samples of interest which for instance
derived from cells or tissues of healthy people and patients suffering on certain
disease phenotypes. Consequently, by applying the strategy of differential
display cloning new genes probably related to a disease phenotype of interest
can be identified directly,thus leading to a new potential drug target [323].
In order to accelerate the process of target identification and target selection
prior to bioassay development and high-throughput screening,chip hybridiza-

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