Recombinant DNA Technology Flashcards
What is recombinant DNA technology?
Involves the transfer of fragments of DNA from one organism or species to another.
Organisms containing transformed DNA known as transgenic
Name three methods in which a DNA fragment can be produced.
Gene machine
Using reverse transcriptase
Using restriction endonucleases
How is reverse transcriptase used to produced desired DNA fragment?
mRNA complementary to the target gene is isolated using ultracentrifugation and is miser with free DNA nucleotides and reverse transcriptase
Reverse transcriptase uses mRNA of target gene as template to synthesise cDNA
How do restriction endonucleases work?
Each type of restriction endonuclease cuts DNA double strand at specific sequence of bases called a recognition sequence.
Recognition sequence is often a specific palindromic sequence that the enzyme recognises and attaches to as the shape is complementary to its active site.
Cuts and digests the DNA at these places.
How is a gene machine used to produced desired DNA fragment?
A database containing necessary information to produce DNA fragments used to synthesise fragments of DNA without the need for a pre existing DNA template
Any sequence of nucleotides can be produced in a short time free of introns.
How is restriction endonucleases used to produced desired DNA fragment?
If recognition sequences present on either side of the target DNA fragment, specific restriction endonucleases incubated with the DNA sample.
Cuts target DNA fragment out by hydrolysis, sometimes leaves sticky ends which is a small tail of unpaired bases on either side of the DNA fragment.
- can be used to anneal the DNA fragment to another piece of DNA with sticky ends of complementary sequences.
Describe the in-vivo method of amplifying DNA. 2 stages
To make the recombinant DNA:
- a vector is used to transfer the DNA can be a plasmid or a bacteriophage
- vector is cut upen using same restriction endonuclease used to isolate target DNA fragment, means sticky ends are complementary
- vector DNA and fragment mixed with DNA ligand which sticks the sticky ends together
Transforming cells:
- vector with the recombinant DNA is used to transfer genes into host cells
E.g host bacterial cells places in cold calcium chloride solution to make cell wall as more permeable and mixture is heat shocked to encourage cells to take up transformed plasmids
E.g with bacteriophage it will infect bacterium by injecting its DNA into it integrating the transformed DNA into the bacterial DNA
How are transformed cells identified?
Marker genes can be inserted into the vector at the same time as the gene that is to be cloned meaning any transformed bacterium will contain the marker.
Host cells are grown on agar plate and each cells divides replicating its DNA
- if marker gene is antibiotic resistance cells grown on agar containing antibiotic so only transformed cells grow
Produces many copies of the cloned gene.
How is the transformed host cell made to produce protein coded for by the target gene?
Vector must contain specific promoter and terminator reigons so RNA polymerase can attach and detach and transcribe the gene
What is PCR?
The polymerase chain reaction is where copies of DNA fragments are made outside of a living organism in several stages which are repeated several times.
Describe the stages of PCR.
- Reaction mix is set up containing the DNA sample, free nucleotides, primers and DNA polymerase
- primers are short pieces of DNA that are complementary to the bases at the start of the fragment sample - DNA mixture is heated to 95 degrees to break hydrogen bonds between complementary base pairs on either strand, it is then cooled to 50-65 degrees so primes can anneal to the strands
- Reaction mix heated to 72 degrees which is the optimum temperature for DNA polymerase to work, free DNA nucleotides form hydrogen bonds with complementary bases and phosphodiester bonds catalysed between adjacent
- new complementary strand is formed - Cycle starts again heating to 95 degrees again using all four strands as templates
How are transformed plants produced?
Gene is inserted into a plasmid which is added to a bacterium
Bacterium is used as a vector to get gene into the plant
How are transformed animals produced?
Gene is inserted into early embryo or female egg, if inserted early enough all body cells will contain desired gene
How are the cells which express the gene controlled?
Promoter region only activated in specific cell types as producing the protein in the wrong cells can damage the organism
What are the benefits of recombinant DNA technology?
- agricultural crops can be made to increase yield, more nutrients and resistant to pests
- industrial processes often used enzymes which can be produced by transformed organisms reducing costs
- many drugs and vaccines produced by transformed organisms, quick cheap and large quantities of
What are some concerns about recombinant DNA technology?
