Polymerase Chain ReactionPolymerase Chain ReactionAimsAimsTo understand the process of To understand the process of PCRPCR and its and its usesuses..

Starter - Match each term with its Starter - Match each term with its correct description (work in pairs)correct description (work in pairs)

5 May 2023

What is it?What is it?

AnimationsAnimations

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

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

InformationInformation Polymerase chain reaction enables Polymerase chain reaction enables

large amounts of DNA to be large amounts of DNA to be produced from very small samples produced from very small samples (0.1ml)(0.1ml)

There is a repeating cycle of:There is a repeating cycle of:separationseparation of double DNA strands of double DNA strandssynthesissynthesis of a complementary of a complementary strand for eachstrand for each

What happens?What happens? Sample DNA , nucleotides, DNA primers & Sample DNA , nucleotides, DNA primers &

thermostable DNA polymerase placed in thermostable DNA polymerase placed in PCR machine.PCR machine.

Strands of sample DNA Strands of sample DNA separated separated by by heating to heating to 9595ooCC

Mixture cooled to Mixture cooled to 3737ooCC to allow to allow primers to primers to bindbind..

Mixture heated to Mixture heated to 7272ooC C for for replicationreplication (optimum temp of DNA polymerase) (optimum temp of DNA polymerase)

Cycle repeats many times (~8mins /cycle)Cycle repeats many times (~8mins /cycle)

ProblemsProblems Separation achieved by heating to 95Separation achieved by heating to 95ooC – C –

no suitable no suitable helicasehelicase DNA polymerase can’t work on completely DNA polymerase can’t work on completely

single stranded DNAsingle stranded DNA – double stranded – double stranded regions needed at the start of sequence to regions needed at the start of sequence to be copied:be copied:

primersprimers (short sequences DNA) (short sequences DNA) complementary to bases at start of region complementary to bases at start of region to be copied usedto be copied used

To synthesize primers , base sequence at To synthesize primers , base sequence at start must be knownstart must be known

TTAACGGGGCCCTTTAAA.....…TTTAAACCCGGGTTTTTAACGGGGCCCTTTAAA.....…TTTAAACCCGGGTTT

AATTGCCCCGGGAAATTT.....…AAATTTGGGCCCAAAAATTGCCCCGGGAAATTT.....…AAATTTGGGCCCAAA

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

The first step for PCR would be to The first step for PCR would be to synthesize "primers" of about 20 letters-synthesize "primers" of about 20 letters-longlong

ONE primer exactly like the lower left-ONE primer exactly like the lower left-hand sequence, and ONE primer exactly hand sequence, and ONE primer exactly like the upper right-hand sequence:like the upper right-hand sequence:

TTAACGGGGCCCTTTAAA.....…TTTAAACCCGGGTTTTAACGGGGCCCTTTAAA.....…TTTAAACCCGGGTTAATTGCCCCGGGAAATTTAATTGCCCCGGGAAATTT.......................>.......................>and:and:<........................................<........................................TTTAAACCCGGGTTTTTTAAACCCGGGTTTAATTGCCCCGGGAAATTT........AAATTTGGGCCCAAAAATTGCCCCGGGAAATTT........AAATTTGGGCCCAAA

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

Remember….Remember…. Nucleotides used must be very pure.Nucleotides used must be very pure. DNA must not be contaminated - DNA must not be contaminated -

any foreign DNA would also be any foreign DNA would also be copied….PCR in legal cases in UK copied….PCR in legal cases in UK suspended at presentsuspended at present

Uses of PCRUses of PCR

http://nobelprize.org/nobel_prizes/http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/illpres/chemistry/laureates/1993/illpres/already.htmlalready.html

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