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DNA recombination - Coggle Diagram

- - - - DNA ligase- catalyses formation of covalent bonds between nucleotide end fragements (binds P and OH)
      - DNA polymerase- amplification
      - Restriction enzymes- recognise and cleave 4-8 sites of DNA (can be straight cuts or sticky ends)
  - - - gene encodes peptide to break down B-galactosidase
      - enzyme breaks down X-Gal to form blue dye
      - DNA fragment is cloned adn reading of LacZ is disrupted so no blue dye
- - - - screening the recombinant clones
      - adding linkers to DNA fragments prior to cloning
    - - direct amplification of genes from genomic DNA
      - degenerate primers for the amplification of unknown DNA seq
      - isolation of tagged genes from transgenic organisms
    - - reverse transcriptase PCR
      - real time PCR
  - - - need 2 primers (forward and reverse)
      - forward primer is same as coding strand and reverse is noncoding strand
      - 17-22 bases, equal CG and AT, little areas of base repeats, little secondary structure
  - - - RE sites can be added by PCR at 5' end of primer
- - - - if DNA sequence from homologous gene (i.e. in another species) then alignment can be used to compare sequences and used as a probe to isolate unknown gene
      - Probes can be generated from a protein sequence (degenerate primers are designed where algorithms works out degenerate reverse primers so only one binds)
  - - - they are archives of genetic material which can be replicated, screened for isolated genes
      - Southern Blotting
        
        can be used to check that DNA probe detects the genes of interest
        
        Process
        
        Nucleic acids separated by agarose-gel electrophoresis (by size)
        
        separated nucleic acids are blotted onto nitrocellulose paper by suction of buffer through gel and paper
        
        the DNA Is immobilised and denatured since treated with alkali
        
        labelled ssDNA fragments can be used to probe the blow for cDNA fragments
        
        short radiolabelled ssDNA fragments are produced and bind to complementary DNA sequences on nitrocellulose filter
        
        Hexamers bind add dATP, dGTP, dTTP and dCTP (where one is radiolabelled) to also bind
        
        radiolabelled probe is hybridised to separate DNA and wait for probe to find band of genomic DNA on membrane
        
        It confirms that a DNA fragment or gene is present in sample of genomic DNA
    - - cleaving of dsDNA with RE
      - inserting this into plasmids and then introduced to DNA bacteria
      - Screening
        
        Disc of membrane is laid on top of bacterial colonies and a few adhere
        
        the cells are disrupted adn DNA denatured and fixed to filter
        
        labelled probe hybridises with cDNA and excess washed off
        
        overlaid X-ray film detects probe and compared to membrane to reveal whihc colonies have required DNA
      - Failings
        
        If the genome is large then so will be the library so many more colonies will have to be screened
        
        some genes are very large because they contain many introns and may be difficult to clone (so use phage libraries)
        
        if hte gene is to be expressed in bacteria but is in a eukaryote and contain introns then the bacteria cannot remove it so no protein
    - - synthesis of cDNA
        
        isolate mRNA and add an oligo(dT) primer (rRNA and tRNA don't have polyA tails so primer cannot bind)
        
        strand synthesis by reverse transcriptase
        
        ribonuclease H degrades most of the RNA
        
        second strand synthesis by DNA pol I
      - Cloning
        
        cloning steps same as gDNA
        
        cloning a gene including promoter and terminator regions can be used to find when and where it is expressed using a reporter gene
      - Screening
        
        the expression vector is transformed into bacterial colonies (these have promoter and terminators so can be expressed)
        
        transfer colonies to a replica plate and lyse bacteria to expose proteins
        
        transfer proteins to nitrocellulose sheet
        
        add radiolabelled antibody specific for protein of interest
      - PCR can be performed by adding MMLV RT to RNA, so mRNA acts as template to produce cDNA then normal PCR