Analysis of Gene Expression

Analysis of Gene Expression

Tapeshwar Yadav(Lecturer)BMLT, DNHE,

M.Sc. Medical Biochemistry

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

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:

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.

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.

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.]

B. Analysis of proteinsThe 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.

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

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.

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.

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.]

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.