RECOMBINANT DNA TECHNOLOGY

involves the transfer of fragments of DNA from one organism or species to another.

restriction endonucleases recognise specific palindrome sequences and will cut DNA at these places

gene machine

the sequence that is required is designed

the first nucleotide in the sequence is fixed to support

nucleotides are added step by step, in the correct order, in a cycle of processes that includes adding protecting groups.

IN VITRO AND IN VIVO CLONING

IN VIVO

insertion - DNA fragments is inserted into a host

transformation - host cell takes up a plasmid and the DNA

identification - shows which cells have been transformed

INSERTION -

uses a vector to transfer the DNA fragment into the host.

vector - plasmid found in bacteria

plasmid - circular DNA found in bacteria

step one - the vector is isolated

step two - the vector DNA is cut open using the same ‘restriction endonuclease’ that was used to isolate the DNA fragment containing the target gene

step three - this produces ‘sticky ends’ of the vector DNA which are complementary to the sticky ends of the DNA fragment containing the gene.

step four - the vector DNA and the DNA fragment are mixed together with the DNA ligase. DNA ligase joins the sticky ends together. this process is called ‘ligation’

IN VITRO

step 2 - annealing the primers

• cooled to between 50 and 60c

• hydrogen bonds are reformed

• oligonucleotides bind to template DNA

• oligonucleotides are called primers as enzymes in step 3 must attach to one of them before it can start to work

• free DNA nucleotide are also added

step 3 - synthesis of new DNA