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Manipulating Genomes (Genetic Engineering (Putting Vectors into Cells…

- - - - Reverse transcriptase is used to find cDNA
      - Primers and DNA polymerase can create double stranded DNA
      - mRNA is known
    - - A DNA probe locates gene
      - Gene can be cut by restriction enzymes
    - - Known sequence can be amplified by PCR
  - - - Alternating periods of hot (42C) and cold (0C) in CaCl2
      - Cell walls and membranes become more permeable and take in the recombinant vector
    - - High voltage pulse disrupts the membrane
    - - Electrical fields to introduce DNA into cells
    - - DNA packaged into bacteriaophage which transfects the host cell
    - - Gold/ Tungsten is coated with the DNA and shot into the cells
- - - - They can then be separated by mass
      - In some cases, proteins can be separated by mass and then, without SDS, according to their surface charge
      - Sodium dodecul sulfate (SDS) is added with the proteins to equalise surface charge
  - - - Primers can be fixed onto a surface
      - The DNA anneals to any complimentary primer, revealing specific genes (e.g. mutated alleles)
- - - - A machine base on Sanger's method was created, using florescent dye
      - The sequence is read off autoradiograms
    - - DNA is cut into fragments then degraded to single-stranded DNA (ssDNA), they are immobilised template strands
      - A sequencing primer is added and DNA incubated with DNA polymerase and other enzymes. Either ATP, TTP, CTP or GTP (activated nucleotides) is added at one time (a nucleotide with 2 extra phosporyl groups)
      - Unincorporated activated nucleotides are degraded and the reaction starts again with another nucleotide
      - It is fast and cheap
      - Once an activated nucleotide is added, it releases the PPi (multiple reactions take place) and gives off visible light, with the intensity correlating with the number of activated nucleotides added in one go
- - - - Number of STRs vary from person to person
      - STR sequences are separated by electrophoresis
      - STRs are polymorphic, however is is extremely rare that people share the same STR sequence
- - - - Alleles can be packaged into liposomes and inserted into the body
      - They may pass through the cell membrane and through the nuclear envelope to insert the genome, which will then be expressed
      - Treatment needs to be repeated regularly
    - - Alleles inserted in viruses which have been modified to be harmless can enter human cells
      - Disadvantages
        
        May provoke immune response
        
        Patient may become immune
        
        Allele may disrupt gene regulating cell division, increasing cancer risk
        
        May be inserted into location which disrupts expression of other genes