Molecular biology

Polymerase chain reactions

this is experimental technique used to make very large amounts of target DNA sequence in vitro. it allows DNA to become aplified several billion fold, effectively purifying the DNA away from the rest of the genome. DNA polymerase uses a single stranded DNA template to 'prime' DNA synthesis. simultaneously priming of both DNA strands at the edges of desired target sequenced by adding and annealing 2 specific primers. DNA is then synthesised using a DNA polymerase. 2 primers are different which are designed to be complementary with target DNA at different locations. it is necessary to have primers of at least 20 bases to ensure specificity.

3 steps of PCR
1. denaturation: denature double stranded DNA to single strands at 95 degree celcius.
2.Annealing: anneal primers to the single stranded DNA at 55 degree celcius
3. extension: DNA is extended by DNA polymerase in 5' to 3' direction at 60 degree celcius.

each step takes about 1 minute, thus one complete cycle takes about 3 minutes.

PCR reactions now have been automated using Taq polymerase. this means enzyme is only added at the beginning and not after every each denaturation step which is very laborious. cycles will continue provided that in the reaction there are sufficient primers and nucleotides.

application of PCR
1. cloning: to isolate genes whose genes are wholly or partially known. after cycles, isolate PCR products from a gel by cutting out the amplified fragments and ligate into vector. no need to screen the library.
2. detect rare events; for example the presence of rare virally infected cells or low abundance pathogens in sample
3. for molecular evolution studies
4. useful in forensic science to detect crime, CSI!
5. diagnose genetic disorder such as Duchenne Muscular dystrophy (musle degenerates)-multiplex pcr products



DNA repair
intro: cell possess systems which can recognise mismatches and structural distortions in DNA

2 main causes of cell damage: physical and chemical agents e.g. UV; errors in DNA replication

2 general classes of DNA damage: single base changes; structural distortions

5 repair systems:
1. direct repair: photoactivation, deoxyribopyrimidine

2.mismatch repair: uracil mismatched, uracil DNA glycosidase (UDG), DNA Pol 1 and DNA ligase, Mut S, Mut L, Mut H, Mut U

3. excision repair: very short, shor or long patch. enzyme UvrABC, UvrD, Dna Pol 1, DNA ligase

4. Tolerance repair: translesion systhesis polymerase (TSP), in e.coli polymerase IV and V, dinB, UmuC, UmuD, medical interest: xeroderma pigmentosum

5. retrieval repair: best repair, use other repair systems, SOS response, lexA, lexA box, SulA, cell division