Class notes
Polymerase Chain Reaction
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overview of steps involved within PCR
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PCR. allows you to amplify DNA sequences
Allows the detection of tiny quantities of DNA in a vast quantity of other DNA e.g. forensic
science
‘The ability to find DNA of interest in the midst of vast contamination’ Femi 2018
Materials and Methods
1) Two primers are needed (forward and reverse) For DNA polymerase to work, a
primer must be present. Why is this? The primer needs to be complementary to the
DNA sequence of interest.
2) PCR machine, heats and cools sample according to the protocol (temperature
instructions are inputted before start).
3) Mix DNA with the two primers, a thermostable DNA polymerase will then be added. 4
DNTPs should be added. A suitable buffer should be used. An excess of primers
should be used (more than the amount of template).
4) Sample is heated (94 degrees) to denature the double stranded template. Once
heating the sample is cooled (50 degrees), so the primers can anneal to the single
strand.
5) Taq polymerase extends the primer (forward) (72 degrees)
6) Cyan polymerase extends the reverse primer
The product becomes the substrate for the next strand, allows continuous amplification.
Each cycle theoretically doubles the number of strands. (for each dsDNA template molecule,
2N double stranded product molecules)
Once cycles have been complete, run the DNA on gel electrophoresis. Use southern blots to
identify if DNA of interest is present. (right size and the right sequence)
Clone the DNA, using restriction enzymes and placing into vectors.
. TOPO cloning (new protocol) This method allows no cutting of the DNA ends.
DNA sequencing
Finding the order of bases/amino acids to understand DNA/RNA molecules
Methods of sequencing DNA, RNA and proteins
. RNA is usually copied into cDNA first using reverse transcriptase.
Proteins sequencing now, are usually identified by working out the DNA sequence within
them.
Sanger sequencing- dideoxynucleotide ‘chain termination’
Begin with a sample of DNA, need to make template DNA single stranded using heat.
This make it available.
Add a short ssDNA primer. Anneals when sample has been cooled. The primer is extended
by the addition of DNA polymerase. (a fluorescent/ radioactively labelled nucleotide to
visualise growing chain)
Stop extension using a dideoxynucleotide triphosphate. Stop the extension in different
places by using different triphosphates. (at different T positions)
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