recombinant dna technology

recombinant dna

introduction of a foreign gene into the dna of another organism . contain the dna from 2 types of organism

genetically modified organism

contains the dna from 2 organisms

what are the 5 steps of making a protein using dna technology

1 - isolation

2 - insertion

3 - transformation

4- identification

5- growth/cloning

why does dna produce the same protein

dna is universal . transcription and translation are universal

isolation of dna fragment - reverse transcriptase dna from mrna

• the mrna has been transcribed from the gene of interest

• reverse transcriptase is used to synthesise a single strand of complementary dna from the mrna molecule

• dna polymerase then forms the other strand of dna from free nucleotides - double strand

advantages of using mrna

• quantity lots of mrna to extract

• searching -stimulate the gene of interest

• the genetic code - proteome is easier - no introns

isolation of dna fragments - restriction endonucleases

• are enzymes found in bacteria that cut dna at specefic base sequences

• can be used to cut out a desired gene from rest of genome

• different restriction enzymes can cut dna into - blunt or sticky ends

sticky ends

• cut ends of DNA

• one strand is longer than the other

• have a strand of single stranded dna

• can attach to complementary dna bases

• will join with another sticky end but only if cut by same restriction endonuclease

restriction endonucleases

• have highly specific active sites that catalyse the hydrolysis of the sugar phosphate backbone of both strands of dna molecule

• cut dna at recognition sites between 4-8 base pairs long

• recogintion sites are palindromic meaning sequence and complement are the same but reversed

• recognition site is defined as a short specific palindromic base sequence

why might some recognition sequences not be useful

occurs within a gene as well as not just near the ends

isolation of dna fragments - gene machine

• desired nucleotide sequence derived from desired protein

• nucleotide sequence fed into computer and oligonucleotides designed = small , overlapping , single strands which form complete gene

• oligonucleotides are created and joined to make the gene

• the gene is replicated many times using pcr . which makes DNA double stranded

• using sticky ends gene may be inserted into a bacterial plasmid which acts as a vector

advantages of gene machine

• fast

• any sequence can be produced

• high accurate

• can remove introns so can be transcribed and translated in prokaryotes

plasmid

a circular piece of DNA seperate from main bacterial DNA

promoter

length of DNA added before the DNA fragment to which transcriptional factors and rna polymerase can bind to intiate transcription

terminator

length of DNA added after the dna fragment which causes RNA polymerase to be released and stops transcription

insertion of genes into plasmids

by inserting gene into a plasmid the plasmid acts as a carrier or vector which can then be introduced back into a bacterial cell

introducing vectors into host cells (transformation)

• plasmids are mixed with bacterial cells in an ice cold medium containing calcium ions

• heat shock (42c) is applied for 2 minutes the bacterial cell walls / membranes increase in permeability allowing the plasmids to pass through into the cell

• only a few bacterial cells will take up the the plasmids. so we need to identify bacterial cells that took this up.

identification using gene markers

• antibiotic resistance markers - works on the principle that a small number of bacteria have two genes that provide resistance to two different antibiotics in the same plasmid

• fluorescent markers - insert the gene for the a fluorescent protein into the plasmid that also has the gene - insert your gene of interest into the centre of a gene for a fluorescent protein - transfer the plasmids into bacterial cells and grow in agar - any bacterial cells that took up plasmids will not be killed

• enzyme markers - there is a particular substrate that is usually colourless but turns blue when lactase acts upon it - insert your chosen gene into the gene containing lactase - transfer plasmids into bacterial cells - grow in agar containing medium and the colourless substrate - any colonies that have taken up the substrate will not turn blue - colourless spots will identify areas that have been transformed