PCR Flashcards
PCR summary
Technique used for selective amplification of DNA in vitro i.e. in tube in lab
Very specific - can amplify specific sequence from mixture
Doesn’t actually purify but makes so much one piece that everything else minor contaminant
Not just cloning - pCR used for lots things e.g. DNA analysis etc
PCR=DNA replication
DNA
- Nucleotides
- BP
- Double-stranded
- Antiparallel
- Complementary
DNA replication
- Semi-conservative
- Need a template strand
- Need a primer
- Polymerase
Overview PCR
One cycle PCR = dissociate the two strands and copy both, 2 molecules of DNA produced
Next cycle do the same again
Each cycle doubling of DNA molecule = exponential
After each cycle - molecules of DNA = multiple by 2 to power of number of cycles
What you need for PCR
A template, can be anything (total DNA from cell/ purified plasmid)
Copies DNA - DNA polymerase
DNA polymerases can’t just copy DNA - need free 3’ -OH to start from, in cells this is an RNA primer, in vitro use DNA primers (easy to make, more stable)
Consequence = we need to know what our sequence is
DNA polymerase needs building blocks to make new DNA strand
Different enzymes for PCR - exact buffer composition may vary but MgCl2 required as an enzyme cofactor
Temperature - each cycle has stages that need specific temperature
Use thermocycler to do PCR as it changes temperature for you
What happens in each cycle
Cycle consists of three stages:
- Denaturation - need a high temperature, double stranded DNA dissociates into single-stranded DNA
- Primer annealing - temp. depends on primer, lower than denaturation, primers bind to complementary sequence of ssDNA, primer binding is antiparallel
- Primer extension - temp depends on enzyme, new strand DNA made, each new dsDNA has one old and one new DNA strand
This is just one cycle - repeated ~ 30 times
Newly synthesised DNA is template for subsequent cycle
Protein stability during PCR
Cycle goes through some high temperatures - 95 oC
DNA polymerases are proteins - usually destroyed by these temperatures
Very first PCR (Kary Mullis, 1983) - added more polymerase during each cycle
Now we have identified thermostable polymerase from thermophilic organisms
DNA polymerases used in PCR
Taq was discovered first, Taq and Pfu are among most common and widely used
Different advantages/disadvantages of different polymerase - choice somewhat dependent on application
Thermostability is important
Extension rate - how fast it can replicate a template
Processivity - how often it falls off and has to re-associate
Proof-reading and fidelity - contribute to accuracy. Pfu introduces fewer mutations into its PCR products
Taq
Thermus aquaticus
Advantages:
- Good thermostability
- Rapid extension rate (~2-4 kb/min)
- High processivity (efficiency)
Disadvantages:
- No proff-reading activity
- Low fidelity (accuracy)
- Adds a 3’ A overhang
Pfu
Pyrococcus furiosus
Advantages
- Superior thermostability
- Slower extension rate (~1 kb/min)
- Low(er) processivity
Disadvantages:
- 3’-5’ exonuclease activity
- High fidelity (accuracy)
- Products are blunt-ended
important features of PCR primers
Have to be complementary to sequence you want to amplify, specific to template
Primers come in pairs, one of each pair will bind to top and one to bottom strand DNA, in opposite orientations (3’ end point towards each other)
So will amplify region of DNA between primers
Always around 20 bp in size (117 bp is the minimum)
Melting temperature (Tm)
Temperature at which primer will dissociate from DNA template
Usually design primers to have Tm≈ 60-64 oC
Primer Tm determines what annealing temperature (Ta) to use in your PCR cycle - should be about 5oC lower than Tm
Probably most important feature of primers
Tm = temp. at which 50% primer is annealed to DNA and 50% is not
Tm and Ta
Ta is just right: primers will bind to your specific sequence
Ta is too low: primers may bind non-specifically to other DNA sequences
Ta is too high: primers may not bind efficiently (or at all), reducing product yield
Old-fashioned method = 2+4: Add 4 for every G/C add 2 for every A/T
Nowadays, we tend to use programmes to calculate Tm for us
Computer programmes also better at spotting things like accidental complementarity (don’t want primer to bind to itself or its pair) or secondary structure
Directly cloning your PCR product
Some issues with ligating a PCR product directly into a vector:
1. Taq adds a 3’ A overhang to its products
- Can remove it
- Clever vectors that use the A overhang - TA cloning
2. PCR products have no 5’ phosphate
- Fine if your vector has a 5’ phosphate
- But then your vector will self-ligate
3. Blunt-ended cloning is not very efficient
4. Blunt-ended cloning is not directional
Taq - can remove A overhang/clever vectors that use the A overhang in clonning
No 5’ phosphate (primers don’t have 5’ phosphorylate) if vector has 5’ phosphate it will self-ligate
Remove vector phosphate and phosphorylate PCR product
Inefficient - have to screen a lot
Insert may be wrong way round
Incorporating restriction sites into primers
Forward primer binds 5’-3’ from start to sto codon
Reverse primer binds 3’-5’, from stop to start codon
Restriction enzymes always added on 5’ end from both forwards and reverse
In this case, forward = EcoRI, reverse = BamHI
Primer site not bound to DNA template
Results in PCR product with restriction sites at each end - can digest as normal then clone
Advantages
- Sticky ends ligate more efficiently
-Directional (use two different enzymes)
- Don’t need to worry about 5’ phosphate/ 3’ Oh on vector
Reverse Transcription PCR (RT-PCR)
Overview:
- RNA is reverse transcribed into DNA (called complementary DNA or cDNA)
PCR used to amplify specific cDNA sequence
Potential uses:
- Molecular cloning of a protein coding cDNA sequence (cloning using mRNA as it doesn’t have introns)
- Analysis of mRNA expression
