Genetic Engineering
Reaction Endonucleases
Nucleases break down polynucleotides
Breaks DNA molecules into smaller fragments so we can study it
"Cuts" at specific sites - not randomly
Break phosphodiester bonds
Genetic Engineering is the transfer of genes from one organism into another organism
The genetic code is universal which means all genes can be inserted, transcribed and translated in any organism
Vector = agent which carries something from one organisme to another
Process
Extract mRNA from pancreas cells
Use reverse transcriptase to make cDNA
Identify insulin cDNA using probes
Add sticky ends to cDNA (GGG)
Methods of isolating genes
Using reverse transcriptase
Cutting out the gene using restriction endonuclease
Making the gene
Use amino acid sequence to determine the DNA base sequence
Computer builds oligonucleotides which are added together to bulld the gene
Gene copied by PCR and given sticky ends
Remove mRNA from cell expressing the gene
mRNA + reverse transcriptase = single stranded cDNA
cDNA + DNA polymerase = double stranded cDNA
Plasmid = small circular DNA molecule found in DNA
Preparation of a vector
Remove plasmid with antibiotic resistance gene and promoter + terminator from bacteria by dissolving bacteria cell wall then centrifuging and extracting plasmid
Cut up plasmid using restriction endonuclease
Add sticky ends (CCC)
Promoter = turns gene on, this is where RNA polymerase binds (allows transcription) these can be sensitive to certain environments so expression can be controlled
Terminator = this is where RNA polymerase finishes transcription
Insertion of insulin gene into vector
Add gene and plasmid together
Sticky ends join up (C-G)
Add DNA ligase which joins up nucleotide backbone
Recombinant DNA (rDNA) made
Isolating recombinant/transformed bacteria
Insert plasmid into bateria - incubate bacteria with plasmid
Plate bacteria onto an antibiotic medium
Only recombinant bacteria survive
Encouraging bacteria to take up plasmid
Plasmid + cells
Add calcium salts
Cool to 0 degrees
Heat to 40 degrees
The bateria become stressed
Replica plating
Technique for testing the genetic characteristics of bacterial colonies
Bacteria is first spread in a petri dish on a medium to support the growth of all bacteria - this is the master plate
A sterile velvet pad, the same size as the petri dish, is then pressed onto the dish, picking up a sample of each colony
The bacteria can then be stamped onto new plates, in the identical arrangement
The medium in the plates contains antibiotics - either one or two different ones
So colonies can be identified that are antibiotic resistant - therefore the desired bacteria can be isolated