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Molecular toolkit (Genomic DNA (contains (exons, regulatory sequences,…

- - - - prevents cutting action by adding a methly group CH3 to the base
        
        Adenine
        
        Cytosine
      - can also be used in eukaryotes to
        
        silence genes
        
        prevent transcription
        
        protect DNA from damage
  - - - cloning a gene
      - carry out genetic transformation
    - - Small variations in base sequence can be detected as differences in the size of fragments of DNA produced following digestion by restriction enzymes
        
        can be used to distinguish between different individuals at the DNA level
- - - - 2) mRNA is copied to single stranded DNA forming a DNA/RNA hybrid
        
        the key to forming cDNA is the enzyme Reverse Transcriptase
        
        synthesises a DNA strand complementary to an RNA template
        
        if provided with a primer that is base-paired to the RNA and has a free 3' - OH group
        
        which the enzyme uses as the starting material and dNTPS
        
        3) The RNA strand is removed using RNAse H and replaced with DNA using a DNA polymerase and dNTPs
- - - - short sequences of single stranded DNA, which are complementary to the regions flanking the region of DNA to be amplified
    - - an enzyme capable of withstanding high temperatures
    - - dATP, dGTP, dTTP, dCTP
    - - The double stranded template-DNA is heated to 92oC
        
        denaturing
        
        On slightly cooling the primers are now able to recognise and bind to the complementary sequences
        
        annealing
        
        There is a forward primer designed to bind at the beginning of the target region and a reverse primer which recognises the end of the target region
        
        temp depends on the primer sequences and tends to be between 45 and 65oC
- - - - an electric current is passed through the gel
        
        DNA has an overall negative charge
        
        DNA molecules are attracted through the gel towards the positive electrode (the anode)
        
        the gel is then removed and stained
        
        separated bands can be seen
        
        smaller DNA fragments move more quickly through the gel than larger molecules
        
        the size of the DNA fragments can be estimated using size markers ( fragments of a known size which are run as a control alongside the DNA samples)
  - - - The capillary paper acts as a wick and transfers “transfer” buffer from the reservoir at the base of the apparatus upwards in to the gel
        
        A positively charged nylon membrane is placed on top of the gel
        
        more damp filter papers followed by paper towels are placed on top of the membrane
        
        The buffer is drawn upwards into the towels, carrying the DNA out of the gel and onto the membrane
        
        The negatively charged DNA sticks to the membrane, importantly conserving the position of the DNA fragments
        
        Once transferred on to the membrane, the identity of the DNA fragments can be investigated further using DNA hybridisation techniques
- - - - 2 main features
        
        the DNA sequence is known
        
        the DNA sequence is labelled
        
        allows the probe to be detected
        
        Radio-labelled nucleotides contain the radioactive isotope 32P and are commonly used
        
        . A labelled probe can recognise and hybridise to the complementary sequence (or target sequence) on the fixed on the membrane
        
        The excess probe is washed away and the membrane incubated with x-ray film
        
        it is possible to identify the position of a particular fragment of DNA in relation to the original gel
- - - - 5' carbon group on the incoming sugar
      - 3' hydroxyl group on the growing chain
  - - - all tubes contain
        
        template
        
        forward primers
        
        deoxynucleotides
        
        (dNTPS) (dATP, dCTP, dGTP, dCTP)
        
        buffer
        
        polymerase
        
        each tube also contains 1 type of chain terminating nucleotide
        
        ddNTP - dideoxynucleotide
        
        does not have a 3' hydroxyl group
        
        prevents further DNA chain elongation
      - the 4 tubes are loaded onto a sequencing gel which separates DNA fragments depending on how many nucleotides are in the chain
- - - - Distribution
        
        achieved by drying / freezing and grinding into a powder
      - Lysis
        
        breakdown of cellular membranes using a detergent
        
        DNA / chromosomal materials are released
    - - typically involves the digestion of proteins with proteinase K
        
        also degrades nuclease which would break down the isolated DNA
        
        proteins are precipitated out of solution using high concentrations of salts such as potassium acetate or ammonium acetate
        
        precipitates are removed by centrifugation
        
        Salting out
        
        Alternative organic extraction method
        
        uses organic substances such as phenol, chloroform and isoamyl alcohol
        
        The correct pH and salt concentration help to ensure the cellular contaminants enter the organic phase and the DNA remains in the aqueous phase
        
        uses toxic compounds and generates toxic waste
    - - usually achieved by Alcohol precipitation
        
        solution is then centrifuged again
        
        the pellet of DNA is recovered and resuspended in either water or Tris EDTA (TE) buffer