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Recombinant DNA and Restriction Enzymes - Coggle Diagram
Recombinant DNA and Restriction Enzymes
Recombinant DNA (rDNA) technology
Techniques used to manipulate nucleic acids
DNA is chemically identical in all organisms
Manipulate DNA/RNA using
Natural biochemical processes
Enzymes
Natural properties of microorganisms
Biochemistry
Microbiology
Techniques
Cut + paste (splice) sequences
Clone sequences
Engineer sequences
Sequence DNA/RNA fragments
Locate fragments/sequences using probe hybridisation
Bacteria as 'workhorses' of molecular biology
Cheap
Easy to manipulate + contain
Exponential clonal growth
Yield is limited by
Nutrients
Space
Naturally move DNA by
Conjugation
DNA transfer
Transformation
DNA uptake
Transduction
Phage transfer
Natural plasmids hosts + replicators
Cloning
General approach
Cut vector
Insert DNA fragment
Insert vector with fragment into bacterium
Vectors have selective markers
e.g. antibiotic resistance
Only bacteria that take up vector will grow
Nucleases
Endonucleases
Cut internally
Specific
Exonucleases
Start at 5' or 3' end
Restriction enzyme classification
Restriction + methylation
Cleaves remotely
Endonuclease or restriction
Cleaves at/close to recognition site
Most widely used
Restriction + methylation
Cleaves close to recognition site
Restriction enzyme nomenclature
HindIII
H - Genus
in - species
d- strain
III - 3rd discovered
RE substrate
Recognition sites
Sequence specificity between enzymes
4,6 or 8 nt long
Some exceptions
Often palindromic
Reads the same on both complementary strands of DNA
Blunt cuts or sticky ends
Zipping together nucleic acids
Ligases
Catalyse ATP-driven ligation of nucleic acid backbones
How many RE sites per fragment/genome?
For DNA sequence length \(y\) nucleotides
The no. of possible sequences \(N_{total}\)
\(N_{total}=x^y\)
In which \(x\) is no. of bases i.e. 4
Example
\(y=6\)
\(N_{total}=x^y=4^6=4096\)
For 100kb genome
Expect to find on avg
\(\frac{100000}{4096}=24.4=24\) EcoR1 sites
Somatostatin
Hormone
Inhibits HGH, insulin, glucagon
14nt long
Known amino acid sequence
E.coli production is unlimited
Plasmid (pBR322)
Codes 2 antibiotics (amp, tet)
Insert synthesised somatostatin gene
Recombinant plasmid