Gene Chips

Dr. Fazli Wahid

COMSATS Institute of Information Technology, Abbottabad

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Gene Chips

Although sequence data provide a profile of all the genes present

in a genome, they give no information as to which genes are switched
on (transcribed) and, hence, which are functionally active at any
given time/under any given circumstances.
Gene transcription results in the production of RNA, either
mRNA (usually subsequently translated into a polypeptide) or rRNA or
tRNA (which have catalytic or structural functions).
The study of under which circumstances an RNA species is
expressed/not expressed in the cell/organism can provide clues as to the
biological function of the RNA (or, in the case of mRNA, the function of
the final polypeptide product).
Furthermore, in the context of drug lead/target discovery, the
conditions under which a specific mRNA is produced can also point to
putative biopharmaceuticals/drug targets.

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Gene Chips

For example, if a particular mRNA is only produced by a cancer

cell, that mRNA (or, more commonly, its polypeptide product) may
represent a good target for a novel anti-cancer drug.
Levels of RNA (usually specific mRNAs) in a cell can be
measured by well-established techniques such as northern blot
analysis or by PCR analysis.
However, the recent advent of DNA microarray technology has
converted the identification and measurement of specific mRNAs
(or other RNAs if required) into a ‘high-throughput’ process.
DNA arrays are also termed oligonucleotide arrays, gene chip
arrays or, simply, chips.

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Gene Chips

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Gene Chips

The technique is based upon the ability to anchor nucleic acid

sequences (usually DNA based) on plastic/glass surfaces at very high
density.
Standard gridding robots can put on up to 250 000 different short
oligonucleotide probes or 10000 full-length cDNA sequences per
square centimetre of surface.
Probe sequences are generally produced/designed from genome
sequence data; hence, chip production is often referred to as
‘downloading the genome on a chip’.
RNA can be extracted from a cell and probed with the chip.
Any complementary RNA sequences present will hybridize with the
appropriate immobilized chip sequence .
Hybridization is detectable as the RNA species are first labelled.
Hybridization patterns obviously yield critical information
regarding gene expression.

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Proteomics

Although virtually all drug targets are protein based, the inference
that protein expression levels can be accurately (if indirectly)
detected/measured via DNA array technology is a false one, as:
1.mRNA concentrations do not always directly correlate with the
concentration of the mRNA encoded polypeptide;
2.a significant proportion of eukaryote mRNAs undergo differential
splicing and, therefore, can yield more than one polypeptide product

Additionally, the cellular location at which the resultant polypeptide will

function often cannot be predicted from RNA delection/sequences nor can
detailed information regarding how the polypeptide product’s functional
activity will be regulated (e.g. via post-translational mechanisms such as
phosphorylation, partial proteolysis, etc.).

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Proteomics

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Proteomics

Therefore, protein-based drug leads/targets are often more

successfully identified by direct examination of the expressed protein
complement of the cell, i.e. its proteome.
Like the transcriptome (total cellular RNA content), and in
contrast to the genome, the proteome is not static, with changes in
cellular conditions triggering changes in cellular protein
profiles/concentrations.
This field of study is termed proteomics.

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Proteomics

Proteomics, therefore, is closely aligned to functional genomics

and entails the systematic and comprehensive analysis of the proteins
expressed in the cell and their function.
Classical proteomic studies generally entailed initial extraction of
the total protein content from the target cell/tissue, followed by
separation of the proteins therein using two-dimensional
electrophoresis.
Isolated protein ‘spots’ could then be eluted from the
electrophoretic gel and subjected to further analysis; mainly to Edman
degradation, in order to generate partial amino acid sequence data.
The sequence data could then be used to interrogate protein
sequence databanks in order to, for example, assign putative
function by sequence homology searches.

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Proteomics

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Proteomics

Two-dimensional electrophoresis, however, is generally capable

of resolving no more than 2000 different proteins, and proteins
expressed at low levels may not be detected at all if their gel
concentration is below the (protein) staining threshold.
The latter point can be particularly significant in the context of
drug/target identification, as most such targets are likely to be
kinases and other regulatory proteins that are generally expressed within
cells at very low levels.
More recently, high-resolution chromatographic techniques
(particularly reverse-phase and ion exchanged-based high- performance
liquid chromatography (HPLC)) have been applied in the
separation of proteome proteins and high-resolution mass
spectrometry is being employed to aid high-throughput sequence
determination.

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COMSATS Institute of Information Technology, Abbottabad

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