Gene expression *The transcription involves synthesis of an RNA from the DNA template and an enzyme called RNA polymerase. *In prokaryotes there is a single RNA polymerase, but in eukaryotes there are three types of RNA polymerase (I,II and III). *Stages of transcription include (I) DNA/RNA polymerase binding (II) chain initation, (III) chain elongation and (IV) chain termination and relase of RNA.

*The relase of RNA molecule may be immediately available for translation (as in prokaryotes) or processed and exported to the cytoplasm (as in eukaryotes). Translation requires a mRNA molecule a supply of charges t RNA (associated with amino acid residues) and ribosomes (r RNA and ribosomal proteins)

*The ribosome acts as “Jig” which holds the mRNA so that the codons may be matched up with the appropriate anti-codon on the t RNA, thus ensuring that the correct amino acid (a.a) is inserted into the growing polypeptide chain. Fig 8

Isolation of DNA and RNA **The first step in any isolation protocol is the disruption of the cell, which may be viral, bacteria, plant or animal. *The method used to open cells should be as gentle as possible to avoid the danger of mechanically shearing large DNA molecules.

* Following cell disruption, a deproteinization step is required. *This is may be achieved by one or more extractions using phenol or phenol/chloroform mixtures. * On the formation of an emulsion and subsequent centrifugation to separate the phases, protein molecules partition into the phenol phase at the interface.

*The nucleic acids remain mostly in the upper aqueous phase, and may be precipitated using isopropanol or ethanol. *If a DNA preparation is required, the enzymatic ribonuclease (RNase) can be used to digest RNA. *If mRNA is needed, a further purification can be performed by oligo(dt)-cellulose to bind the poly (A) tail of eukaryotic mRNA (Fig 9)

* For preparing plasmid DNA (PDNA), a technique of gradient ultracentrifugation is often used A cesium chloride (Cscl) solution containing DNA is spun at high speed over a long period, a density gradient is formed and a band of DNA is then taken off.

Handling and quantification of nucleic acids The concentration of a solution of nucleic acid can be determined by measuring the absorbance at 260 nanometers (nm), using spectrophotometer.The concentration of a solution of nucleic acid can be determined by measuring the absorbance at 260 nanometers (nm), using spectrophotometer. *An A260 of 1.0 is equivalent to a concentration of 50 mg/ml for double- stranded DNA, or 40 mg/ml for single- stranded DNA or RNA.

The ration A260/A280 indicates if there are contaminants present. The ration should be 1.8 for pure DNA and 2.0 for pure RNA. * In the presence of UV- absorbing contaminants, which makes spectrophotometric measurements impossible, DNA concentration is determined by the fluorescence of ethidium bromide dye.The ration A260/A280 indicates if there are contaminants present. The ration should be 1.8 for pure DNA and 2.0 for pure RNA. * In the presence of UV- absorbing contaminants, which makes spectrophotometric measurements impossible, DNA concentration is determined by the fluorescence of ethidium bromide dye.

The dye intercalates between the DNA bases and fluoresces orange when illuminated with UV light. Precipitation of nucleic acids is an essential technique, which may achieved by adding ethanol or isopropanol to a DNA solution in a ration by volume of 2:1 in the presence of 0.2M salts. After precipitation the DNA is recovered by centrifugation, the pellet can be dried and the nucleic acid re-suspended in the buffer.The dye intercalates between the DNA bases and fluoresces orange when illuminated with UV light. Precipitation of nucleic acids is an essential technique, which may achieved by adding ethanol or isopropanol to a DNA solution in a ration by volume of 2:1 in the presence of 0.2M salts. After precipitation the DNA is recovered by centrifugation, the pellet can be dried and the nucleic acid re-suspended in the buffer.

Radiolabelling of nucleic acids Labelling the nucleic acid with a radioactive molecule (usually deoxynucleoside triphosphate (dNTP), labelled with 3 H or 32 p), enable keeping track of the small amounts of nucleic acid in any cloning procedure.Labelling the nucleic acid with a radioactive molecule (usually deoxynucleoside triphosphate (dNTP), labelled with 3 H or 32 p), enable keeping track of the small amounts of nucleic acid in any cloning procedure.

*A second application of radiolabelling is the production of high radioactive nucleic acid molecules ( Probes ) for use in hybridization experiments.

Some common methods of labelling are described are described below: (1) End labelling (2) Nick translation (3) Primer extension.Some common methods of labelling are described are described below: (1) End labelling (2) Nick translation (3) Primer extension.