LECTURE - Real Time PCR Flashcards
components of a real-time PCR
- DNA polymerase
- buffer
- magnesium calcium
- nucleotides
- template DNA
- primers
- probes
- passive reference dye
*no agarose gel (endpt); use fluorescence)
Taq DNA polymerase
- thermostable DNA-dependent DNA polymerase which synthesizes a complementary strand of DNA in a 5’ to 3’ direction
- forms phosphodiester bonds between 3’ OH group of a bound nucleotide & 5’ phosphate of an incoming nucleotide
- it has a 5’-3’ exonuclease activity to remove and correct erroneously incorporated nucleotides
real-time PCR primers
- should be designed to amplify a target of 5 0to 150 bp
- should be 18-24 nucleotides in length
- should have ~50% GC
- primers should NOT form secondary structures or hybridize w one another
- melting temp of primer pairs should be within 1 C
- ideally, primers will end with C or G
- many primer design software programs available
multiple types of probes for real time PCR including
- Taqman (hydrolysis probes); 5’ nucleus probes
- FRET
- molecular beacon
- scorpion
passive reference dye
used to normalize the fluorescent signal of reporter dyes
- they provide a stable baseline for multiplex real-time PCR and qRT-PCR
- normalize fluctuation in fluorescence resulting from: pipetting variation, bubbles in rxn well, evap/condensation, machine noise, variations in instrument excitation + detection
Ct
- threshold cycle
- cycle number at which the rxn crosses the threshold
functions of std curves
- take efficiency int account
- convert Ct’s to quantity units
- test dynamic range
100% PCR efficiency
amount of DNA doubles with every cycle
- slope of std curve
- recommended = 90-110%
what can influence PCR efficiency in real-time PCR?
amplicon length structure GC content suboptimal reagent concentrations presence of inhibitors
this detects contamination of PCR reagents
no template control
what does the NTC reaction contain?
all real-time PCR components except the template
what does a positive signal in an NTX reaction indicate?
presence of contaminating nucleic acids
> NTC should always be neg
- sample result is NOT valid if NTC is positive
positive PCR control
- can be a known positive control or an absolute std
- demonstrates rxn is specific for the target
what can an internal positive control be used to test for?
presence of PCR inhibitors
the threshold should fall in the ____________ phase
exponential q
