Polymerase Chain Reaction
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What does 'in vitro' mean?
In a test tube
How is in vitro cloning designed to occur?
In an artificial setting and repeatedly in a laboratory process
When is PCR usually employed?
- When scientists wish to produce large quantities of a particular sequence of DNA from very small quantities
- This is used in genetic fingerprinting for forensic analysis or to amplify small quantities of rare DNA for cloning purposes
What is used during the PCR reaction?
- A mixture of double stranded DNA
- DNA nucleotides
- A heat resistant version of DNA polymerase
- Short lengths of single strand DNA known as primers
To where are primers complementary?
- A sequence before the region of interest on the strand of the template
- Just after the region of interest on the other strand
What are primers used for?
They provide the starting sequence for DNA replication and define the region to be amplified
Describe stage 1 of PCR
- The double stranded DNA is split into 2 single stranded templates by heating to 95oC for 30 seconds
- This breaks the hydrogen bonds between the bases
Describe stage 2 of PCR
- The mixture is cooled to 40oC for 30 seconds
- This allows the primers to hydrogen bond to the complementary bases on the templates
Describe stage 3 of PCR
- The mixture is heated to 72oC for 2 minutes
- Nucleotides with a complementary base hydrogen bond to the template
- This is the optimum temperature for the DNA polymerase enzyme which joins DNA nucleotides together with a phosphodiester bond to build up a new complementary strand alongside the template strand
Describe stage 4 of PCR
The mixture is once again heated to 95oC to separate the newly formed strands and the process is repeated for another 25 cycles (the number of strands doubles after each cycle)
State 3 advantages of in vivo cloning
- There is no risk of contamination as only fragments with the correct sticky ends are inserted into the plasmid. With PCR any contaminating DNA could also be copied
- Bacteria will also produce the protein that the gene codes for
- Much more accurate in copying DNA
Give one advantage of in vitro cloning
It is extremely rapid due to lack of culturing cell needed in vivo
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