Cloning

Vector must be linearized before foreign DNA can be inserted. 

The linearized vectors are treated with alkaline phosphatase to prevent re-ligation. Foreign DNA is then inserted into the plasmid vector by the action of ligase to produce a recombinant molecule 

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  • Created by: Chelsey
  • Created on: 27-03-13 12:49

Cloning

Vector must be linearized before foreign DNA can be inserted. 

The linearized vectors are treated with alkaline phosphatase to prevent re-ligation. Foreign DNA is then inserted into the plasmid vector by the action of ligase to produce a recombinant molecule 

Transformation:

E.coli can be treated with CaCl2 to alter the permeability of cell wall and then heat shocked - high voltage electric pulses induce cell walls to fuse  

Electroporation is an alternative to chemically treated competent cells 

Many Bacillus spp. are naturally competent

Restriction enzymes are a barrier to transformation

Methylation of DNA is a barrier to transfer between species

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Screening for transformants

Alpha - complementation

  • a quick and easy 1 step process of determining whether a transformed bacterial colony 
  • lac - Z gene produce (beta galactosidase) is a tetramer, and each monomer is made of two parts - lacZ alpha and lacZ omega. If the alpha fragment was deleted the omega fragment is non-functional but the alpha fragment functionality an be restore in-trans via plasmid. 
  • neither peptides are active by themselves bu when both are present together, they spntaneously reassemble into a functional enzyme.
  • When DNA fragments are inserted in the vector, the production of lacz alpha is disrupted, and the cells therefore show now beta galactosidase activity. 
  • The presence or absense of an active beta galactosidase can be detected by X-gal which produces a blue dye when cleaved with beta galatosidase.
  •  transformed clones have both parts of the gene (vector contributes missing portion) so blue dye shows
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The Polymerase Chain Reaction

  • Developed in 1983 by Kerry Mullis 
  • Used to amplify DNA fragments and relies on thermal cycling  
  • DNA is denatured by increasing the temperature to 95oC (replaces helicase)
  • The temperature is reduced to around 45 -60oC to allow the primers anneal to the template
  • Temperature is increased to 72oC and the polymerase extends the chain
  • Heat stable enzyme required as high temps result in denaturation
  • Thermus aquaticus is the source of Taq polymerase which is used in PCR

Optimising PCR:

  • Well - designed primers 
  • Adjust MgCl2 concentration (essential cofactor of DNA polymerase, many components in reaction bind magnesium ion e.g. primers and dNTPs)
  • Annealing temperature optimised based on primer Tm (Tm=optimal melting temperature) often 5oC below Tm
  • Type of polymerase (Pfu and VENT have increased fidelity relative to Taq
  • Hot- start prevents annealing and extension at low temps - avoids no specific amplification by inactivating Taq polymerase at low temps 
  • Touch down PCR - primers avoid amplifying nonspecific sequences. Annealing temp determines the specificity of primer annealing. At temps jujst below Tm only very specific pairing between the primer and tmeplate will occur.   
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Variations and applications of PCR

  • Standard PCR (screening, cloning, detection, making probes)
  • Sequencing, Microsatellites (characteriztion of polymorphisms, mapping)
  • Mutagenesis 
  • PCR-RFLP (amplification of sites with known varients - DNA sample is broken into pieces and fragments separated according to lengths)
  • Nested PCR - two pairs of PCR primers are used for a single locus. First pair amplifies locus normally, second pair (nested pair) bind within the first PCR produce and produce a second PCR product that will be shorted that the first. Prevents wrong locus being amplified. 
  • Multiplex PCR - rapidly detects deletions or duplications. Amplifies myltiple targets in a single PCR experiment. More than one target sequence can be amplified by using multiple primer pairs in the reaction mixture 
  • Inverse PCR is used to amplify DNA with only one known sequence. Allows PCR to be carried out even if only one sequence is available from which primers may be designed 
  • PCR primers can introduce desired sequence into amplified DNA - primer sequenced incorporated into amplified DNA and forms template bery soon after first few rounds of amplification 
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PCR and the diagnosis of HIV

  • Antibodies are detectable 3-12 weeks after infection by antibody titres may fall with time
  • ELISA antibody test is useless in new born infants for several months because of maternal IgG antibodies
  • PCR allows amplification of tiny quantities of viral DNA to allow early treatment 
  • Does not rely on the presence of antigens or antibodies in the blood for diagnosis 
  • RNA PCR is used to screen blood donations
  • DNA PCR is used to detect HIV in infants born to mothers known to have the virus 
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Restriction fragment length polymorphisms

  • RFLPs are sequence variants that create or destroy a restriction site in DNA therefore altering the length of the restriction fragment tht is detected - generating different sized bands on Southern blots 
  • They are generated in various ways:
    • loss of an endonuclease cleavage site, most proably as a result of a base change 
    • by insertion or deletion of a stretch of DNA
    • by variation in the number of repeats of a microsatellite
  • It is possible to do RFLP without PCR from circular molecules as they have a defined size and can be isolated selectively by varying pH when precipitating the DNA. mitochondrial DNA have higher mutation rate variability for many population level studies 
  • Draw backs:
    • different mutations can produce same band
    • too many mutations and the entire signal is lost
    • too few mutation and not enough polymorphisms are observed 
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PCR - RFLP for fast cheap diagnosis of haemophilia

  • mutation in clotting factor VIII gene: intron 18
  • codominant - shows heterozygous arriers as well as homozygous sufferer 
  • No need for sequencing - fast and efficient way of detecting variants  
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