MBIO 3410 lecture Nov 15 2007

MBIO 3410 lecture Nov 15 2007 - Last Lecture Overview cDNA...

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1 Last Lecture Overview cDNA Libraries Treatment of cDNA ends Directional cDNA Cloning Fractionating mRNA Enriching mRNA Use of methylated forms of deoxynucleotides Use of rarecutting restriction enzyme Problems associated with oligo-dT primers Gene Identification by Screening DNA Probes Colony and Plaque Nucleic Acid Hybridization
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3 Immunoscreening
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4 Immunoscreening If a DNA fragment library is cloned into an expression vector the gene products encoded within the foreign DNA may be produced within the host cell Mechanisms to identify these unique polypeptide sequences can be used to screen the library in order to identify particular clones The detection of polypeptide sequences is usually performed using antibody screening procedures
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5 Antibodies Antibodies are raised when a foreign protein or peptide is injected into an animal: Rabbit, mouse, sheep, goats, pigs and horses The presence of the foreign protein ( antigen ) is detected in the animal by surface receptors on B and T lymphocyte cells Each B cell has many thousands of different receptors on its surface that are able to bind to particular antigens The binding of the antigen to an individual receptor results, via a complex pathway, in the descendants of that B cell secreting vast numbers of the soluble form of that particular receptor = antibodies
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6 Polyclonal antibodies : are isolated from the serum of an immunized animal and contain many different antibodies that recognize different epitopes of the same antigen Monoclonal antibodies : are produced from isolated, clonal cells and recognize an individual specific epitope within the antigen Antibodies
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7 Antibodies that bind to proteins can recognize either: Continuous epitopes i.e. the primary amino acid sequence of the protein Discontinuous epitopes formed by the folding of the protein to generate a surface area antigen that is composed of different segments of the primary structure The bulk of naturally occurring epitopes are of the discontinuous type Antibodies for use in the laboratory are often produced using denatured protein to ensure that the produced antibodies recognize continuous epitopes and can thus be used to detect the denatured protein using western blotting
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8 Immunoscreening of a
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MBIO 3410 lecture Nov 15 2007 - Last Lecture Overview cDNA...

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