2. AnAlysis of Gene expression
The tools of biotechnology not only allow the
study of gene structure, but also provide ways
of analyzing the products of gene expression -
mRNA and proteins
A.Determination of mRNA levels
B.Analysis of proteins
3. A. DeterminAtion of mrnA
levels
Messenger RNA levels are usually determined
by the hybridization of labelled probes to
either mRNA itself or to cDNA produced from
mRNA.
1.Northern blots:
2.Microarrays:
4. 1.northern blots:
Northern blots are very similar to Southern
blots except that the original sample contains a
mixture of mRNA molecules that are separated
by electrophoresis, then transferred to a
membrane and hybridized to a radioactive
probe.
The bands obtained by autoradiography give a
measure of the amount and size of particular
mRNA molecules in the sample.
5. 2. Microarrays:
DNA microarrays contain thousands of
immobilized DNA sequences organized in an area
no larger than a microscope slide.
These microarrays are used to analyze a sample for
the presence of gene variations or mutations
(genotyping), or to determine the patterns of mRNA
production (gene expression analysis), analyzing
thousands of genes at the same time.
For genotyping analysis, the cellular sample is
genomic DNA.
For expression analysis, the population of mRNA
molecules from a particular cell type is converted to
cDNA and labelled with a fluorescent tag.
6. Contd…
This mixture is then exposed to a gene chip, which is a
glass slide or membrane containing thousands of tiny
spots of DNA, each corresponding to a different gene.
The amount of fluorescence bound to each spot is a
measure of the amount of that particular mRNA in the
sample.
DNA microarrays are often used to determine the
differing patterns of gene expression in two different
types of cell—for example, normal and cancer cells.
[Note: Physicians hope to one day be able to tailor
particular treatment regimens to each cancer patient,
based on the specific microarray expression patterns
exhibited by that patient's individual tumor.]
7.
8.
9. B. AnAlysis of proteins
The kinds and amounts of proteins in cells do not
always directly correspond to the amounts of
mRNA present.
Some mRNA are translated more efficiently than
others, and some proteins undergo
posttranslational modifications by adding sugars
or lipids, or both.
Thus, the genome contains 20,00 to 30,000
genes, but a typical cell produces hundreds of
thousands of distinct proteins.
10. Contd…
When investigating one, or a limited number of
gene products, it is convenient to use labelled
antibodies to detect and quantify specific proteins.
However, when analyzing the abundance and
interactions of large numbers of cellular proteins
(called proteomics), automated methods
employing two-dimensional gel electrophoresis,
mass spectrometry, multidimensional liquid
chromatography, and bioinformatics are
employed
11. 1.enzyme-linked immunosorBent AssAys
(elisA):
These assays are performed in the wells of a plastic
microtiter dish.
The antigen (protein) is bound to the plastic of the
dish.
The probe used consists of an antibody specific for
the particular protein to be measured.
The antibody is covalently bound to an enzyme,
which will produce a colored product when exposed
to its substrate.
The amount of color produced can be used to
determine the amount of protein (or antibody) in the
sample to be tested.
12. 2. Western Blots:
Western blots (also called immunoblots) are
similar to Southern blots, except that protein
molecules in the sample are separated by
electrophoresis and blotted (transferred) to a
membrane.
The probe is a labeled antibody, which
produces a band at the location of its antigen.
13. 3. Detecting exposure to HiV:
ELISA and Western blots are commonly used to detect
exposure to HIV by measuring the amount of anti-HIV
antibodies present in a patient's blood sample.
ELISA are used as the primary screening tool, because
they are very sensitive.
These assays sometimes give false positives, however,
so Western blots, which are more specific, are often
used as a confirmatory test .
[Note: ELISA and Western blots can only detect HIV
exposure after anti-HIV antibodies appear in the
bloodstream. PCR-based testing for HIV is more useful
in the first few months after exposure.]
14. 4. proteomics:
Involves the study of all proteins expressed by a
genome, including their relative abundance,
distribution, posttranslational modifications, functions,
and interactions with other macromolecules.
The 20,000 to 30,000 genes of the human genome
translate into hundreds of thousands of proteins when
alternate splicing and posttranslational modifications
are considered.
While a genome remains unchanged, the amounts and
types of proteins in any particular cell change
dramatically as genes are turned on and off.
Proteomics offers the potential of identifying new
disease markers and drug targets.
