Recombinant DNA Technology Flashcards
(33 cards)
Explain 3 pharmaceutical uses of recombinant dna tech
Human insulin for diabetes
Factor VIII for haemophilia
Hepatitis B vaccine produced antigens
Name the 4 cells used potentially in dna recombination
Bacteria cells - prokaryotes host cells
Yeast cells - eukaryotic
Insect cells - eukaryotic
Mammalian cells - eukaryotic
Name the 4 enzymes involved in dna technology
Restriction endonucleases - cleaves dna fragments
Reverse transcriptase - turns mrna for gene into cDNA
Taq polymerase - used in PCR
Dna ligase - synthesise the 2 strands together in vector and injected dna
Why do restriction endonucleases originate from bacteria
Bacteria use it to cleave phage dna which infects bacteria
Why are restriction endonucleases called specific
They cleave dna at specific sites in DNA called palindromic sequences (same base sequence on 2 strands)
Why do restrictionenzymes recognise palindromic sequences
They are 2 homodimers (2 proteins) which are identical so on each strand identify same sequence
What are the 2 ways DNA sequences are cut my restriction enzymes
Symmetrical cleavage - blunt ends
Asymmetrical cleavage - sticky ends used by dna ligase
What is the process called where dna ligase will join the dna fragments and the plasmid dna where it has been cut by SAME enzyme
Annealing
What are 3 things vectors must have to be used in recombinant dna tech
Unique restriction sites where they can be cleaved
Oric- where dna is cloned
Gene to allow selection (identifying the recombinant hosts) - this can be eg the Lac Z gene for enzyme B galactosidase
Plasmids are the most common vector, explain why they are used
They replicate independantly and quick in bacterial
They have genes such as Ampicillin resistance to allow selection
They have restriction sites
Some have expression factors (able to express a gene into a protein)
Bacteriophages are also vectors. How are they used
The dna fragments are inserted into them so when they inject bacteria, they inject the dna fragment
This then joins the plasmid (PROPHAGE)
What are phagemids and why are they good vectors
Hybrids where plasmids are contained in phages - larger fragment can be cloned
To isolate random DNA sequences there are 2 techniques - reverse transcriptase or using restriction endonucleases, explain both
Restriction enzymes are used to cleave random dna into fragments when extracted from cells
Reverse transcriptase uses mrna strands eg isolated from genes for eg insulin
Then transcribes it into cDNA (double stranded)
How are specific fragments isolated from dna using PCR
Dna strands are hybridised with primers specific to that base sequence (strands are seperated)
These are then cloned many times to produce many fragments of the same DNA sequence
When dna fragments are inserted then into plasmids via dna ligase and restriction endonucleases (same ones), explain how the bacteria is then transformed to accept the recombinant plasmid
The bacteria goes through HEAT SHOCK AND CACL is added (calcium chloride)
This forms pores in the bacteria membrane
The pores allow diffusion of the plasmid into the bacteria where it is kept on ice
When warmed to 37C the cell fixed the cell wall and bacteria are grown on agar plates
Why is the plasmid kept on ice to transform the bacteria
Prevent cell growth - because cell wall is damaged/pores the bacteria would undergo apoptosis and die
How is antibiotic Eg ampicillin resistance used to select the hosts with insert plasmids
The insert hosts will have ampicillin resistance in the plasmid so when added to the medium they survive and wild type don’t
What is an issue with using the antibiotic resistance principle to select inserts
Some bacteria will have turned the plasmid back into normal plasmid when joined by dna ligase - they would still have the amp resistance gene
How is INSERTIONAL INACTIVATION used to select for host inserts
The dna fragment can be inserted into genes such as the Lac Z gene which produces B galactosidase
If the fragment is inserted it inactivated the b galactosidase
Can’t digest X gal into blue pigment
Blue pigment won’t show up in inserts
What checks can be made to see if the dna wanted is in the host
Using dna probes which only recognise the base sequence
What are the 3 ways recombinant dna is used after gene tech
1- to express the gene into proteins
2- recover plasmids and manipulate them - by isolating from bacteria cells
3- transfer into useful mamalian cells
What do plasmids need to be able to induce expression of the protein
An expressive factor - they need promoter sequences next to the gene to allow transcription and translation
Why are 2 restriction endonucleases needed to cut the plasmids
To allow for the right orientation of the DNA sequence - allowing expression of the gene
Advantages of using bacterial cells as hosts
Simple
Short generation time
Large yield of product
