W5 Gene Expression Analysis Flashcards
essential components in pcr reaction
thermostable dna polymerase (usually taq)
forward and reverse primers specific to target of interest
dNTPs
divalent cations: MgCl2 > forms complexes with dNTPs to be substrates for taq polymerase and stabilise primer to dna
PCR reaction buffer containing Trust-Cl to maintain pH
template dna (sample)
why is optimal annealing temperature required
too high > primers anneal poorly to template
too low > nonspecific annealing of primers
optimal T: usually 3-5 degrees lower than Tm of oligonucleotide primers
conditions for extension of primers
performed at or near optimal temperature for dna synthesis
extension time normally 1 min per kb of product
conditions for standard PCRs catalysed by Taq polymerase
25-30 cycles
annealing conditions: 45-60 degrees depending on calculated Tm of primer pair
extension conditions: 68-70 degrees
properties primers should have
length of 18-24 bases
40-60% G/C content
start and end with 1-2 G/C pairs
melting temperature of 50-60 degrees
primer pairs should have Tm within 5 degrees of each other
primer pairs should not have complementary regions
what is RT-PCR (reverse-transcription pcr)
used to detect presence of ran by converting into cdna > amplification of cdna
semi quantitative as results seen at endpoint, not real time > pcr product stop increasing exponentially at end point as reacting components are limiting > this method more of js qualitative
useful for detecting rare transcripts or analysis of samples which are in limiting amounts
what is RT-qPCR (reverse transcription quantitative pcr)
allows amplification and quantification of RNA
real time detection during each cycle using fluorescent dye or probe, which increases proportionally to amount of dna amplified
step 1 of RT-PCR
- denaturation of rna and primers at 70 degrees for 5 min to remove secondary structures
- first strand cdna synthesis: incubation of rna template with enzyme and reaction mix, rna primers > performed at 42 degrees for 1 hr to achieve max cdna yield and length
step 2 of RT-PCR
amplification of specific genes via pcr
no RT control must be included to check for contaminating dna in sample
final concentration of 0.2 micrometers for each primer pair
extension step using hot start taq polymerase performed at 65 to 68 degrees with extension rate of 1 min per kb
in RT-PCR, what are the requirements for designing primers to prevent amplifying genomic dna instead of cdna
primer spans and exon-intron boundary > primer cannot bind if got intron
primers flank an intron
if primers cannot be designed to separate exons or exon-exon boundaries > must treat rna sample with RNase-free DNase I or dsDNase to remove contaminating genomic dna
how to set up real time quantitative PCR
cdna sample and pcr master mix prepared
fluorescent label added to pcr mix prior to starting the reaction
how quickly the amplified target reaches a threshold detection level correlates with the amount of starting material present
change in fluorescence over course of reaction measured
types of fluorescent dyes for real time q-pcr
non-specific dna binding agents: SYBR green I
sequence specific dna probes: TaqMan probe
how does SYBR green I work
dye only fluoresces when bund to dsDNA
dna denatured > dye released and fluorescence reduced > during extension primers aneal and pcr product (dsDNA) generated > increase in binding of dye to more dna > increase in fluorescence
how does TaqMan probe work
fluorescent reporter (R) dye and quencher (Q) attached to 5’ and 3’ ends of Taqman probe
when probe is intact, reporter dye is quenched > during extension, dna polymerase cleaves the reporter dye from probe > released reporter dye emits fluorescence once polymerisation completed
what is fold change
the difference in dna copies between samples
fold change = 2 ^ (Ct1- Ct2)
higher amount of starting template > lower ct
