3) Amplifying DNA (PCR)

Question 1

“Ingredients” for PCR

Answer 1

• Template DNA (the sequence to be amplified)
• DNA PRIMER (chemically generated oligonucleotides) - to start the synthesis of new DNA strand
• dNTPs (equal concs of A,T,G,C)
• Polymerase to copy template (Thermostable Taq polymerase is used, obtained from thermus aquaticus)
• Buffer (Tris-HCl) to maintain pH and provide necessary salt conc
• MgCl2 - essential for polymerase activity. Conc of Mg2+ ions affects stringency of primer binding

Question 2

Denaturation step

Answer 2

• dsDNA is denatured by heating to 95C

- target sequence is made accessible

Question 3

Annealing step

Answer 3

• temp is cooled to 40-50C
• Precise temp is critical and each PCR system has to be defined and optimised. (primers have different annealing temps. Always go for the lower temp, but further away from optimum temp = less specific primer binding).
• Reactions that aren’t optimised –> other DNA products in addition to specific targets (or no amplification at all)
• This step allows primers (which are present in excess), to bind to their complementary sites. They provide a free 3’ end for DNA polymerase to work on.

Question 4

Extension/elongation step

Answer 4

• temp is increased to 72C, optimum temp for Taq pol to replicate template DNA
• PCR is run for 2 hours (30 cycles)
• after each cycle, amount of DNA increases exponentially
• Once all the cycles are complete, the temp is reduced to 4C to stop reactions

Question 5

PCR primer design

Answer 5

• Primers shouldn’t be able to anneal to themselves
• they should have similar G/C content so that the annealing temps will be similar
• Primers must anneal to opposite strands for exponential replication to occur

Question 6

RT-PCR

Answer 6

• for the first cycle, PCR is used to convert ssRNA into cDNA (using a primer and reverse transcriptase)
• The cDNA is then amplified through PCR, and the rate of amplification is proportional to the amount of cDNA (and therefore RNA) that you started with.
• If the the product produced is measured after every cycle, we can accurately measure the amount of DNA/RNA we started with

Question 7

Applications of PCR

Answer 7

• genetic testing
• personalised medicine
• Diagnosis of DNA
• Forensic DNA
• probes for southern/northern blots
• prepare samples for sequencing
• sequence ancient DNA
• surveying microbiomes
• measuring gene expression
• RT-PCR
• qPCR