PCR Flashcards
What’s PCR & E.g
a method used to amplify DNA from small amount to large
e.g. ID genetic mutations, forensic testing
What must you do /not do when doing PCR?q
- Wear PPE
- Clean tubes & pipette tips
- Don’t talk over tubes
- Minimise touching things w/ gloved hands
- Separate areas for different stages of PCR procedure
PCR theory (in vitro)
- DNA denature @ 95ºC = disrupt H bonds => dsDNA -> ssDNA
- Annealing @ ~55ºC = primers bind to ssDNA
- Extension @ 72ºC, DNA polymerase extends complementary strand - add dNTP @ 3’ OH of primer
- steps repeated in many cycles
PCR components?
- reaction buffer
- (MgCl2) > require Mg 2+ as a cofactor for Taq polymerase
- hpH2O
- All dNTPS
- a pair of primers
- DNA to be amplified
- Taq (DNA polymerase) [most expensive]
What types of controls you may need to set up for a PCR?
- Pos. control: sample contains same region of interest to be amplified = should be 1 band > checks for validity
- *Neg. : contains all components except DNA > ensures reagents are not contaminated OR check DNA is not amplified non-specifically
- Amplification: uses a 2nd set of primers - not specific to region of interest but are specific for a region in sample (for reference) > ensures you have the sample in the rxn & rxn works (important for neg. controls)
Typical reation volume for PCR is
20-50uL
What do you do if your positive control is not working or has multiple bands?
- Invalid
- Perform PCR again
- Check expiry dates
What do you do if your neg control is not working or has multiple bands?
Everything you prepped is invalid = throw everything away & start again
After PCR what is usually done? & why
Run on gel to check:
• if you have PCR products
• expected PCR product => then can be used for sequencing, cloning, restriction digest
typical PCR program
- initial denaturation
- denaturation
- Annealing
- Extension
- Reapeat steps 2-4 in cycles
- Final extension > ensure all PCR product are same length
Factors affecting PCR
- polymerase used: w/ or w/out (incorporate wrong dNTP) proofreading activity
- Primer design
- Annealing temperature
How might [Mg2+] affect PCR?
- if Mg2+ too much = more non-specific products (stringency dec)
- from primers binding non-specifically
- must be Hi enough that’s >[dNTP]
How might the polymerase used affect PCR?
- Taq not have proofreading ability => can insert incorrect dNTP = can cause mutation
- terminal transferase > make sticky overhangs
- pfu & Vent polymerase - polymerase w/ proofreading ability & make blunt ends > for determine SEQUENCE of DNA
Difference b/w FWD primer & RVS primer
- FWD primer: uses RVS template strand => forward strand
- RVS primer: uses FWD template strand => RVS strand
How might the primer design affect PCR?
- primer length = more dNTP = more H+ bonds = bind to self
- nucleic acid composition: G-C have 3x Hbond Hi annealing temp than A-T have 2x Hbond
- Sequence of homology: mismatch of bases = less H bond
what factors must be considered when deigning a Primer
- ~3-5ºC below melting temp of primer of lowest Tm of primer pair
HOw can you determine the primer Tm’s (what equation)
= 4(G+C) + 2(A+T)
*add up bases in sequ.
How can you determine optimal annealing temp.
use thermal cycler w/ gradient PCR: test which temp. allows optimal annealing
Relationship b/w temp & stringency (specific binding)
- low temp = more likely to bind = but binds less specific= lo stringency
- Hi temp = less likely to bind but binds more specific = Hi stringency
What makes an ideal primer?
- 18-25bp long
- has a G-C of ~50% (30-70%) > want try h ave equal C-G & A-T = keep Tm close
- Tm’s ~55-60ºC
- primer’s shouldn’t be complimentary to each other = primer dimers
When setting up a hot start PCR why do we set up on ice?
reduce activity of Taq = mispriming
How is allel-specific PCR used?
- detect SNP mutations
- mismatch @ 3’ end dNTP = won’t amplify
Describe mutagenic PCR or the FDB assay
- have Primers not complementary to target
= intoduce mutations in PCR products
= introduce R. sites
= digested = mutant
Describe isothermal PCR
- use DNA polymerase that can denature DNA while amplifying it (e.g. Bst polymerase)
- PCR rxn occur @ single temp
= lower cost & equipment
describe Loop-mediated isothermal amplification (LAMP)
- DNA initially denatured & chilled
- Bst polymerase added & incubated @ single temp
*uses @ least 4 primers in single rx
= form dumbell structure
= exponential amplification of DNA
What is the approximate Tm of the following primer? 5’-AGCTTACGGATCCAATGCAA-3’ *
~58ºC
You perform a PCR for which you are expecting a single band. Instead, you get multiple bands. Which of the following would potentially solve this issue?
restart w/ new primer (bc maybe there is multiple primers?)
What is the extension rate of a standard Taq polymerase?
~1kb /min
You perform a PCR for which you are expecting a single band. Instead, you get no bands. Which of the following would potentially solve this issue?
restart w/ new reagents
