PCR

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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)

04 September 2015

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What is it?

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Animations

http://www.dnalc.org/ddnalc/resources/pcr.html

http://www.maxanim.com/genetics/PCR/pcr.swf

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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

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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)

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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

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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.

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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

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