Polymerase Chain Reaction

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Presentation transcript:

Polymerase Chain Reaction 06 July 2018 Polymerase Chain Reaction Aims To understand the process of PCR and its uses. Starter - Match each term with its correct description (work in pairs)

What is it?

Animations http://www.dnalc.org/ddnalc/resources/pcr.html http://www.maxanim.com/genetics/PCR/pcr.swf

Information Polymerase chain reaction enables large amounts of DNA to be produced from very small samples (0.1ml) There is a repeating cycle of: separation of double DNA strands synthesis of a complementary strand for each

What happens? Sample DNA , nucleotides, DNA primers & thermostable DNA polymerase placed in PCR machine. Strands of sample DNA separated by heating to 95oC Mixture cooled to 37oC to allow primers to bind. Mixture heated to 72oC for replication (optimum temp of DNA polymerase) Cycle repeats many times (~8mins /cycle)

Problems Separation achieved by heating to 95oC – no suitable helicase DNA polymerase can’t work on completely single stranded DNA – double stranded regions needed at the start of sequence to be copied: primers (short sequences DNA) complementary to bases at start of region to be copied used To synthesize primers , base sequence at start must be known

TTAACGGGGCCCTTTAAA.....…TTTAAACCCGGGTTT AATTGCCCCGGGAAATTT.....…AAATTTGGGCCCAAA the sequence of bases which ONLY flank a particular region of a particular organism's DNA, and NO OTHER ORGANISM'S DNA. This region would be a target sequence for PCR.

The first step for PCR would be to synthesize "primers" of about 20 letters-long ONE primer exactly like the lower left-hand sequence, and ONE primer exactly like the upper right-hand sequence: TTAACGGGGCCCTTTAAA.....…TTTAAACCCGGGTT AATTGCCCCGGGAAATTT.......................> and: <........................................TTTAAACCCGGGTTT AATTGCCCCGGGAAATTT........AAATTTGGGCCCAAA

DNA polymerase must be thermostable (37oC – 95oC used) to avoid fresh enzyme being added.

Remember…. Nucleotides used must be very pure. DNA must not be contaminated - any foreign DNA would also be copied….PCR in legal cases in UK suspended at present

Uses of PCR http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/illpres/already.html

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