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

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Polymerase Chain Reaction Aims  To understand the process of PCR and its uses. Starter - Match each term with its correct description (work in pairs) 16 November 2015

What is it?

Animations s/pcr.html s/pcr.html R/pcr.swf R/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 95 o C  Mixture cooled to 37 o C to allow primers to bind.  Mixture heated to 72 o C for replication (optimum temp of DNA polymerase)  Cycle repeats many times (~8mins /cycle)

Problems  Separation achieved by heating to 95 o C – 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.....…TTTAAACCCGGGTTTAATTGCCCCGGGAAATTT.....…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 (37 o C – 95 o C 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 mistry/laureates/1993/illpres/already.html mistry/laureates/1993/illpres/already.html