Gene expressionGene expression*The transcription involves synthesis of an *The transcription involves synthesis of an RNA from the DNA template and an enzyme RNA from the DNA template and an enzyme called called RNA polymeraseRNA polymerase..

*In prokaryotes there is a single RNA *In prokaryotes there is a single RNA polymerase, but in eukaryotes there are three polymerase, but in eukaryotes there are three types of RNA polymerase types of RNA polymerase (I,II and III).(I,II and III).*Stages of transcription include *Stages of transcription include (I)(I) DNA/RNA DNA/RNA polymerase binding polymerase binding (II)(II) chain initation, chain initation, (III)(III) chain elongation and chain elongation and (IV)(IV) chain termination chain termination and relase of RNA.and relase of RNA.

*The relase of RNA molecule may be *The relase of RNA molecule may be immediately available for translation immediately available for translation (as in prokaryotes) or processed and (as in prokaryotes) or processed and exported to the cytoplasm (as in exported to the cytoplasm (as in eukaryotes).eukaryotes).

Translation requires a mRNA Translation requires a mRNA molecule a supply of charges t RNA molecule a supply of charges t RNA (associated with amino acid (associated with amino acid residues) and ribosomes (r RNA and residues) and ribosomes (r RNA and ribosomal proteins)ribosomal proteins)

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

Isolation of DNA and RNAIsolation of DNA and RNA**The first step in any isolation **The first step in any isolation protocol is the disruption of the cell, protocol is the disruption of the cell, which may be viral, bacteria, plant or which may be viral, bacteria, plant or animal.animal.

*The method used to open cells *The method used to open cells should be as gentle as possible to should be as gentle as possible to avoid the danger of mechanically avoid the danger of mechanically shearing large DNA molecules. shearing large DNA molecules.

**Following cell disruption, a Following cell disruption, a deproteinization step is required.deproteinization step is required.

*This is may be achieved by one or more *This is may be achieved by one or more

extractionextractionss using using phenol or phenol or phenol/chloroform mixtures.phenol/chloroform mixtures.

**OnOn the formation of an emulsion and the formation of an emulsion and subsequent centrifugation to separate the subsequent centrifugation to separate the phases, protein molecules partition into the phases, protein molecules partition into the phenol phase at the interface. phenol phase at the interface.

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

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

Handling and quantification Handling and quantification of nucleic acidsof nucleic acids

•The concentration of a solution of nucleic The concentration of a solution of nucleic acid can be determined by measuring the acid can be determined by measuring the absorbance at absorbance at 260260 nanometers (nm), using nanometers (nm), using spectrophotometer.spectrophotometer.

*An A260 of *An A260 of 1.01.0 is equivalent to a is equivalent to a concentration of concentration of 5050 mg/ml for double- mg/ml for double- stranded DNA, or stranded DNA, or 4040 mg/ml for single- mg/ml for single- stranded DNA or RNA. stranded DNA or RNA.

•The ration The ration A260/A280A260/A280 indicates if indicates if there are contaminants present. The there are contaminants present. The ration should be ration should be 1.81.8 for pure DNA for pure DNA and and 2.02.0 for pure RNA. for pure RNA.* In the presence of UV- absorbing * In the presence of UV- absorbing contaminants, which makes contaminants, which makes spectrophotometric measurements spectrophotometric measurements impossible, DNA concentration is impossible, DNA concentration is determined by the fluorescence of determined by the fluorescence of ethidium bromideethidium bromide dye dye..

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

and the nucleicand the nucleic acid re-suspended in the acid re-suspended in the buffer.buffer.

Radiolabelling of nucleic acidsRadiolabelling of nucleic acids

•Labelling Labelling the nucleic acidthe nucleic acid with a with a radioactive moleculeradioactive molecule (usually (usually deoxynucleoside triphosphate deoxynucleoside triphosphate (dNTP) , labelled with (dNTP) , labelled with 33H or H or 3232p), p), enable keeping track of the small enable keeping track of the small amounts of nucleic acid in any amounts of nucleic acid in any cloning procedure.cloning procedure.

*A second application of *A second application of radiolabelling is the radiolabelling is the production of high radioactive production of high radioactive nucleic acid molecules nucleic acid molecules

((ProbesProbes) for use in ) for use in hybridization experiments.hybridization experiments.

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