PCR Flashcards
What is PCR?
DNA amplification detected by gel electrophoresis or probes
What we need
Template DNA, primers, free dNTPs, Buffer, Polymerase enzyme + MgCl2
3 cycle steps
Denaturation= 95 °C
Annealing = 60°C
EIongation = 72°C
Analysis after PCR
Buffer covers gel, current applied, DNA migrate to +ve electrode
ARMS PCR requires
precise base matching at 3’ end
PCR for SNP
Selective amplification : Primer matches or mis-matches at 31and
ARMS PCR Principle
2 reactions with 3 primers :
1 primer complementary in Both reactions= shows Reaction is working (+ ve control)
others differ at 3’ end for normal or mutant ( 1 in each tube)
match= extension
ARMS Result
Homozygous= Amplification in only 1 tube
Herero= Both tubes
control = always +ve
Blank = always -ve
+ve control requirements
Diff size to products so it is visible on gel
Taq DNA Pol
Lacks 3’ to 5’ exonuclease proofreading activity = doesn’t correct mis-matched primer
why use blank?
shows no contamination
PCR for CF
ARMS PCR (2 arms=2 tubes)
Designing primer
Always 5’ to 3’ direction
calc Size of product
No. Of bases from From start of forward primer to start of reverse Primer
Prevent carryover x4
filter tips+ designated Pipettes, uv irradiation, uracil N glycosylase, Aliquot reagents
Real time PCR Prevent carryover
Sealed reaction+ no post-PCR manipulation
Uni - directional workflow
from clean (pres to dirty (postPCR) = Prevents contamination
UraciI N -glycosylase principle
dUTP used instead of dTTP, enzyme removes uracil = Not used as template
Reverse Transcriptase PCR
measurers MRNA expression :
1) Reverse transcription= RT enzyme produces cDNA,
2) PCR Primers bind target = elongation, gel electrophoresis
oligo dT primers
Specificity to MRNA, allow many targets on CDNA
Random primers
Synthesise large pools of CDNA by annealing throughout, Commonly qPCR
3 PCR graph phases
Exponential : 100%. efficiency (doubling)
Linear: components being used up/ degrading
Plateau : End Point =stopped
q (Real time) PCR Principle
Detection during exponential phase by Reporter fluorescence
signal
Directly proportional to no. of amplicons
qPCR method
same as PCR but with Probes
uses DNA or CDNA
SYBR Green detection
Binds any dSDNA, Product formed= increased fluorescence
real Time disadvantage
No gel run= assume correct DNA
Melt curve analysis
Product slowly heated + sequence determines temp = determines correct DNA
Melt curve results
1 peak= 1 product
incorrect products examples
Non specific products= diff target
Primer dimers (short)
identify by running an gel
Probe (Taqman)
3’ quencher 5’ reporter
Reporter cleaved
Relies on NO exonuIease activity
Real Time probes bind when
During extension
Threshold Cycle ( CT)
Cycle where amount of reporter fluorescence > threshold
lower CT = Higher starting no. of copies
Absolute Quantitation uses
standard curve
Relative Quantitation ( Delta CT)
Differences in expression level of gene between diff samples uses control (housekeeping) gene =fold change
Delta CT
CT (target)- CT ( Ref)
D D CT
D CT (treated sample) - D CT (untreated)
Fold change calc
2 ^ -D D CT
uses of q PCR example
HIV monitoring
