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Recombinant DNA Technologies
Recombinant DNA, genetics studies and bacteria
58
Biochemistry
Undergraduate 1
01/10/2013

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Cards

Term
What is genetic engineering?
Definition
The induced mutation of genes to produce a product that can be harvested
Term
What is cloning?
Definition
Creating a genetically identical copy of a organism
Term
What is recombinant DNA technology?
Definition
Creating, cloning and utilising recombinant DNA
Term
What is needed to do recombinant DNA technology?
Definition
DNA/RNA, enzymes, vectors and cells
Term
What are the four types of cell used in recombinant DNA technologies?
Definition
Bacteria, yeast, insect and mammalian
Term
What are the four enzymes used recombinant DNA technologies?
Definition
Restriction enzymes, DNA ligase, Taq polymerase ad Reverse Transcriptase
Term
What do restriction enzymes do?
Definition
Cut DNA
Term
What does DNA Ligase do?
Definition
Stick fragments of DNA together
Term
What does Taq Polymerase do?
Definition
Alows DNA replication to occur at the high temperatures in PCR
Term
What does reverse transcriptase do?
Definition
Copies RNA into DNA
Term
How did restriction enzymes come about?
Definition
Defense mechanism against bacteriophages
Term
How do restriction enzymes know which section of DNA to cut?
Definition
They recognise the palindromic sequences
Term
What is a palindromic sequence?
Definition
A sequence that is the exact copy on the template strand
Term
What are the two types of cut a restriction enzyme can do?
Definition
Symmetrical and asymmetrical cleavage
Term
What does symmetrical cleavage produce?
Definition
Blunt ends
Term
What does asymmetrical cleavage produce?
Definition
Sticky ends
Term
What are the two types of sticky end?
Definition
5' overhang and 3' overhang
Term
What are the four properties of a good vector?
Definition
1) Has unique restriction sites for DNA insertion
2)Is competent
3)Has an origin of replication
4)Contains a gene that produces a selection factor
Term
What is the most common vector?
Definition
Plasmids
Term
How big are plasmids?
Definition
2,200kb
Term
What are the two other vectors?
Definition
Bacteriophages and cosmids
Term
What is a cosmid?
Definition
A plasmid that can be inserted into bacteriophages
Term
How do we create recombinant DNA?
Definition
Obtain the DNA of interest and insert it into a vector
Term
Where does the DNA in recombinant DNA technologies come from?
Definition
Random fragments of DNA cut up to form a genomic library or a cDNA library
Term
What is a genomic library?
Definition
Lots of fragments of one DNA cut up
Term
What is a cDNA library
Definition
The copy of DNA using reverse transcriptase
Term
How is the DNA amplified?
Definition
By PCR
Term
What does PCR stand for?
Definition
Polymerase Chain reaction
Term
How is the DNA inserted into a vector?
Definition
1)Restriction enzyme cuts ope plasmid
2)Gene is then inserted and DNA ligase then seals up the gap
Term
How do we clone recombinant DNA?
Definition
Vector is inserted into a host, host then divides many times, the many vectors are then purified
Term
What are the two ways that a vector can be inserted into a bacteria?
Definition
Heat shock or electroporationj?
Term
How does heat shock work?
Definition
CaCl2 is added over ice, then they are heated to 42 degrees
Term
How does electroporation?
Definition
A series of electric shocks are administered over the membrane to disrupt it
Term
After the vector is inserted what are the three possible types of cell?
Definition
->Taken up plasmid
->Taken up recombinant plasmid
->Not taken up any plasmid
Term
How are bacteria selected for recombinant DNA?
Definition
Using ampicillin resistence
Term
What type of plasmid is usually used for vectors?
Definition
pUC18
Term
Outline the mechanism of identifying recombinant DNA bacteria with Ampicillin and Tetracyline?
Definition
1)Grow on ampicillin kills off those without plasmid
2)Grow on tetracycline kills off recombinant DNA bacteria
3)Then go back to work out which were killed off on original plate
Term
Outline mechanism of identifying recombinant DNA with the LacZ gene?
Definition
1)Grow on plate containing X-gal
2)Those with LacZ gene (No recombinant DNA) will glow blue as they will break down X-gal
3)Those without LacZ gene (recombinant DNA present) will not glow as they cant break down X-gal
Term
What are the three ways we can identify a colony containing the cloned DNA?
Definition
Complimentary DNA probes, PCR of primers and screening for the expression of a particular protein
Term
How can we use recombinant DNA?
Definition
To express proteins, manufacture the plasmid or transfer the plasmids
Term
What is an expression vector?
Definition
A plasmid specifically made for expressing proteins
Term
What must an expression vector contain as well as the gene?
Definition
A promoter sequence before the gene
Term
What are introns?
Definition
Junk DNA between the exons
Term
What are exons?
Definition
The parts of DNA that make up mRNA
Term
What is splicing?
Definition
The removal of introns
Term
What does pre-mRNA contain?
Definition
Introns and Extrons
Term
What does mRNA contain?
Definition
Exons
Term
What are the four advantages of using bacterial cells?
Definition
1)Simple
2)Fast replication
3)Large yield
4)Low costs
Term
What are the three disadvantages of using bacterial cells?
Definition
1)Eukaryotic proteins can't be folded correctly
2)Proteins can be toxic to bacteria
3)No post-translational modification
Term
What are the four advantages of using yeast cells?
Definition
1)Unicellular eukaryote
2)Similar to mammals
3)Grows quickly and cheaply
4)Perform post-translational modifications
Term
What are the two disadvantages of using yeast cells?
Definition
1)Proteases degrade some proteins
2)What are the four advantages of using bacterial cells?
Term
What are the four advantages of using insect cells?
Definition
1)High level protein expression
2)Correct folding of mammalian proteins
3)Cheaper than mammalian cells
4)Post-translational modifications
Term
What is the disadvantage of using insect cells?
Definition
1)What are the four advantages of using bacterial cells?
Term
What are the three advantages of using mammalian cells?
Definition
1)Produce mammalian proteins
2)Fold proteins correctly
3)Post-translational modifications are correct
Term
What are the three disadvantages of using mammalian cells?
Definition
1)Complexity
2)Lower cell counts
3)Expensive
Term
Name one product or recombinant DNA technologies used commercially?
Definition
Insulin
Term
What are the three problems with using animal insulin?
Definition
1)Side effects of it being different
2)Difficult to purify
3) Possible contamination
Term
How has recombinant DNA technology been used in HIV research?
Definition
CD4 antibody blocks CD4 receptor and HIV cannot infect. Adding the recombinant DNA that blocked the production of the CD4 receptor prevented HIV infection
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