Topic 8—B: Genome projects and gene technologies- 3. Amplifying DNA fragments Flashcards
What will you probably want to do once you’ve got a fragment of DNA?
- you will probably want to make more copies
How is making more copies done?
- this is done by gene cloning
What is gene cloning all about?
- making loads of identical copies of a gene
What are the 2 different techniques of gene cloning?
- in vivo cloning
- in vitro cloning
What is in vivo cloning ?
- where the gene copies are made within a living organism
- as the organism grows and divides, it replicates the DNA, creating multiple copies of the gene
What is in vitro cloning?
- where the gene copies are made outside of a living organism using the polymerase chain reaction (PCR)
Once you have got the DNA fragment containing the target gene, what can you use it for?
In vivo cloning
In vivo cloning stages
Part 1- making recombinant DNA
- the first step in vivo cloning is to insert the DNA fragment into a vectors DNA
- The vector DNA is isolated and then restriction endonucleases and DNA ligase (an enzyme) are used to stick the DNA fragment and vector DNA together
Here’s how it works:
1. The vector DNA is isolated
2. The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene. This means that the sticky ends of the vector DNA are complementary to the sticky ends of the DNA fragment containing the gene
3. The vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA. This process is called ligation
4. The new combination of bases in the DNA (vector DNA + DNA fragment) are called recombinant DNA
Part 2- transforming cells
- the vector with the recombinant DNA is used to transfer the gene into cells (called host cells). Host cells that take up the vectors containing the gene of interest are said to be transformed. If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA
- with a bacteriophage vector, the bacteriophage will infect the host bacterium by injecting into it
- The phage DNA (with the target gene in it) then integrates into the bacterial DNA
Part 3- identifying transformed cells
- only around 5% of host cells will take up the vector and its DNA so its important to be able to identify which cells have been transformed.
- marker genes can be used to identify the transformed cells
1. Marker genes can be inserted into vectors at the same time as the gene to be cloned.
- this means any transformed host cells will contain the gene to be cloned and the marker gene
2. Host cells are grown on agar plates and each cell divides and replicates its DNA, creating a colony of cloned cells
- Transformd cells will produce colonies where all the cells contain the cloned gene and the marker gene
- the marker gene can code for antibiotic resistance- host cells are grown on agar plates containing the specific antibiotic so only transformed cells that have the marker gene will survive and grow
- or the marker gene can code for fluorescence- when the agar plate is placed under a UV light only transformed cells will fluoresce
3. Identified transformed cells are allowed to grow more, producing lots and lots of copies of the cloned gene
What is vectors DNA?
- a vector is something that’s used to transfer DNA into a cell
- vectors can be plasmids (small, circular molecules of DNA in bacteria) or bacteriophages (viruses that infect bacteria)
If you want the transformed host cells to produce the protein coded for by the DNA fragment what will you need to make sure?
- that the vector contains specific promotor and terminator regions
What are promotor regions?
- they are DNA sequences that tell the enzyme RNA polymerase where to start producing the mRNA
What are terminator regions?
- they tell it where to stop
What will happen without the right promotor region?
- The DNA fragment wont be transcribed by the host cell and a protein wont be made
Where may the promotor and terminator regions be present?
- in the vector dna or they may have to be added in along with the fragment
What can PCR be used to make?
- millions of copies of a fragment of DNA in just a few hours
In vitro cloning (stages)
- A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase
- the DNA mixture is heated to 95 degrees to break the hydrogen bonds between the 2 strands of DNA
- The mixture is then cooled to between 50 degrees and 65 degrees so that the primers can bind (anneal) to the strands - The reaction mixture is heated to 72 degrees so DNA polymerase can work.
- The DNA polymerase lines up free DNA nucleotides alongside each template strand and joins the nucleotide strands together
- specific base pairing means new complementary strands are formed - 2 new copies of the fragment of DNA are formed and one cycle of PCR is complete
- The cycle starts again- the mixture is heated to 95 degrees and this time all 4 strands (2 original and 2 new) are used as templates
What are primers?
They are short pieces of DNA that are complementary to the bases at the start of the fragment you want
What is DNA polymerase?
- It’s an enzyme that creates new DNA strands
What does each PCR cycle double?
The amount of DNA
