Techniques 2: PCR Flashcards
What do we mean when we say ‘PCR is exponential’?
the molecules double each cycle
How many cycles is typical for PCR?
30
What do we need for PCR?
- template strand
- DNA polymerase
- Primers
- dNTPs (deoxyribonucleotide triphosphates)
- Buffer (MgCl2)
- Thermocycler
Describe the first stage of PCR
Denaturation - 95OC
= Double stranded DNA dissociates into single stranded
Describe the second stage of PCR
Primer Annealing - 55-65OC
Primers bind to complementary sequence on ssDNA
Primer binding is antiparallel
Describe the 3rd stage of PCR
Primer extension - 68-72OC
DNA polymerase synthesises new strands of DNA from the 3’ end of the primers
Why is Taq polymerase suited to the high temps required in PCR?
Because it originates from a bacteria living in aquatic thermal vents = optimum temp is high
What is the melting point in PCR?
Temp when the primer dissociates from DNA template
Usually design primers have a Tm ~60-64C
Primer Tm determines what annealing temp Ta to use in the PCR cycle → 5OC lower than the Tm
What happens in PCR if the annealing temp is too low?
primers bind to non-specifically to other DNA sequences
What happens in PCR if the annealing temp is too high?
primers may not bind effectively (or at all) reducing product yield
Which direction does a forward primer amplify in?
5’-3’ (same as transcription)
Which direction does a reverse primer amplify in?
3’-5’
What is reverse transcription PCR (RT-PCR)?
makes a cDNA from an mRNA sequence
What are the potential uses of RT-PCR?
Molecular cloning of a protein coding cDNA sequence
Analysis of mRNA expression
Describe cDNA synthesis in RT-PCR
First strand:
1.Reverse transcriptase synthesis of the first cDNA strand
2. Poly(dT) primers bind poly(A) tail of the mRNA
3.mRNA is removed
Second strand:
1. Synthesised by the Klenow fragment of DNA polymerase I
2.The hairpin formed by RT acts as a primer
3.The ss-DNA loop can be digested by a nuclease
