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