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Genetic engineering

Genetic engineering (tools, methods, techniques, molecular level) is one aspect of genetic modification (changing gene)

Genetic engineering- isolate DNA and insert into existing cells to modify an organism to change one of its characteristics



• Guide RNA signals where the Cas9 enzyme cuts the DNA
• Cas9 nuclease (bacterial enzyme) unwinds and cuts both strands of DNA at a specific location
• Gaps repaired, knock in or knock out to repair faulty alleles and restore their normal function, or disable the gene STOP signal


Restriction enzymes

• Cut DNA using restriction enzymes (breaks covalent bonds and phosphate backbone* (active site is complementary to base sequence, cuts and isolates the sequence, found naturally in bacteria)
○ Blunt ends-base pairs and sticky ends- one chain is shorter than the other, exposes nitrogenous base overhanging (hydrogen bonds broken)

○ Recognise, bind to and cut specific sequences of DNA



Electrophoresis is used to separate genes according to their lengths (shorter lengths move faster and farther)
move towards the positive electrode
shorter lengths travel faster and farther


DNA ligase

joins DNA fragments, forms covalent bonds



• Molecular probe can be used to identify fragments, have to denature DNA fragments first, probe attaches to complementary strand, radioactively labelled


DNA fragments made?

DNA fragments can be artificially made (DNA synthesisers join nucleotides, up to 100 bp)
DNA fragments can also be made by using an mRNA template



PCR can be used to amplify DNA fragments
• Denaturation- breaking H bonds to create two single stranded molecules from one double stranded, at 94 degrees Celsius
• Annealing- Primers (single stranded DNA) attach (upstream), starting points 55 degrees Celsius
• Extension- using the primers as a starting point, DNA (taq) polymerase attaches complementary nucleotides to the chain, 72 degrees Celsius



Plasmids- circular DNA naturally found in bacteria that store antibiotic resistance genes, then used to transport foreign DNA
Identical between daughter genes
Plasmids are used as vectors and can be swapped among themselves
Origin of replication- DNA sequence where DNA replication begins
Selectable marker gene- antibiotic resistance gene codes for proteins that prevents antibiotic from killing the cell, used to distinguish those bacteria which the foreign gene integrated successfully into
Multiple cloning site- recognition sites for restriction enzymes, sticky ends


Recombinant plasmids

Recombinant plasmids are made through cutting the plasmid's DNA and the foreign/desired DNA base sequence by a restriction enzyme, mixed (form weak hydrogen bonds), ligase is added so covalent bonds are formed

Electroporation- cell placed in an electric field, creates holes in pm, allows desired plasmid to enter bacterial cell
Heat-shock- ice-cold solution that includes recombinant cells and bacteria-> 42 degree Celsius, fluidity of pm, increased chances of recombinant plasmid being taken up-> antibiotic kills any bacteria that didn't take up the recombinant plasmid