Summary
MBY364 Theme 1 summary
- Course
- MBY364 (MBY364)
- Institution
- University Of Pretoria (UP)
Full summary of theme 1 (restriction and ligation of DNA molecules) made using lecture notes, the textbook and class notes.
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RESTRICTION AND LIGATION OF DNA MOLECULESSTEPS OF GENE CLONING
1. Identification of a source from which the target gene can be isolated and
preparation of the gene as passenger DNA
2. Selection of a suitable DNA vector and its preparation as a carrier molecule
3. Ligation of the passenger DNA to the vector DNA by a ligase enzyme
4. Insertion or transfer of the recombinant molecules into competent host cells
by means of transformation
5. Screening and selection of the recipient cells that contains the recombinant
DNA molecules
NB: Treatment with CaCl2
- The cells are treated with CaCl2 and subject to heat shock
- This allows the DNA to enter the cell
- CaCl2 destabilises the membrane and the heat shock creates pores through
which the DNA can enter
- Ca (+) attracts DNA (-) and acts as a carrier molecule to bring DNA into the
bacterial cell
- Heat shock is 42°C for E. coli
, DNA
MANIPULATION ENZYMES
- There are 4 broad categories of manipulation enzymes
- Nucleases - enzymes which cut, shorten or degrade DNA
- Exonucleases - cleave the DNA from the ends
- Endonucleases - cleave the DNA from within (typically used to degrade
DNA)
- Restriction endonucleases - cut DNA at specific sequences (used for
cloning as the ends generated are predictable and reproducible)
- Ligases - join DNA molecules together
- Create a phosphodiester bond between two adjacent nucleotides in the
DNA backbone
- Discontinuity repair - break in one strand of DNA is repaired
- Joining two molecules - ends of two separate DNA molecules are
joined together
- Polymerases - copying of nucleic acid templates
- DNA polymerase I - has nuclease and polymerase activity
- Klenow fragment - has 3’-5’ exonuclease and 5’-3’ polymerase activity
to fill single stranded nicks
- The Klenow fragment is a fragment of Pol I without nuclease activity
- Both are referred to as DNA dependent DNA polymerases - use a DNA
template to synthesise DNA
- Reverse transcriptase - synthesises DNA from an RNA template
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