qpcr
qpcr Quantitative PCR or Real-time PCR Gives a measurement of PCR product at end of each cycle real time Differs from endpoint PCR gel on last cycle Used to determines relative amount of template DNA at start of PCR. More sensitive than end-point PCR using a gel
Applications of qpcr How much of the DNA of interest is in there? RT-qPCR: Compare levels of gene expression between two samples. Forensics, food safety: Precisely how much of the DNA of interest, and pathogen, is present? Gene copy number: amplified genes in cancer Viral load in patient
Phases of PCR Cycle number Taken from Biotechniques without permission; http://www.biotechniques.com/biotechniquesjournal/specialissues/2008/april/twenty-fiveyears-of-quantitative-pcr-for-gene-expression-analysis/biotechniques-45217.html?pagenum=2
Principle of quantitation for real-time Two samples: A has 1 copy B has 4 copies PCR Threshold cycle (Ct) is 16 copies ØWhat cycle does each cross the threshold? Do the calculation. ØTry it again if B has 8 copies.
Principle of quantitation for real-time PCR The difference in DNA copies between the samples is 2 CtA CtB = 2 DCt àassumes 100% efficiency for each cycle
Plot [DNA] on log scale Fluorescence (RFU) 10-fold dilution series Taken from the following website without permission; https://www.promega.com/resources/pubhub/getting-thebest-results-from-your-reverse-transcription-quantitative-pcr-assay/
Exponential phase of PCR All reagents at optimal concentration Reaction is 100% efficient, all templates are copied Rate is dependent on amount of template Can compare amount of product in two samples with confidence.
Linear phase of PCR Reagents decrease below optimal concentration, used in reactions. Not all templates copied Efficiency is different between samples cannot rely on the measured values of product
Plateau phase of PCR Reaction winding down, reagents depleted, product inhibition End point, samples for gel taken here Amount of product at plateau not proportional to the amount of DNA at the start Sample with low template at start can catch up to sample with high template.
PCR qpcr PCR detects product at end point, gel No quantification Limited range of detection, what you can see on a gel (0.5 to 5.0 ng/band) Determine size and # of products qpcr detects product at each cycle, in real time Measures amount of product at end of each cycle More sensitive than PCR, <100 copies of product Cannot determine size, but can determine # of products
Range of detection Detection of PCR product PCR detects product at end point, gel qpcr detects product at each cycle, in real time
Detection of product SYBR Green 1. SYBR Green I dye binds to double-stranded DNA (dsdna). 2. SYBR Green I dye binds to each new copy of dsdna during PCR. 3. As amount of dsdna increases in successive round of PCR, fluorescence intensity increases Fluorescence intensity is proportional to the amount of PCR product produced.
SYBR Green binds to dsdna SYBR Green binding depends on: - length of product - amount of A/T Does not saturate DNA - cannot correlate fluorescence w/length PCR Unbound SYBR Green I Bound SYBR Green I Fig. 2.1. DNA-binding dyes in real-time PCR. Fluorescence dramatically increases when the dye molecules bind to dsdna.
What is causing SYBR Green signal? Desired PCR product Incorrect product if present Primer-dimers Not single stranded DNA
How many dsdnas are in reaction? Each unique dsdna should have a unique melting temperature when is splits into ssdna. Melting depends on length and nucleotide sequence The melt curve is required when using SYBR green. Generated after final cycle of qpcr Peak on curve = melting temperature (50% of molecules are melted)
Melt curve analysis one dsdna little/no decrease in RFU, no melting Decrease in RFU, melting Decrease in RFU slows as [dsdna] decreases Decrease in fluorescence with change in temperature: -d(rfu)/dt Where would you expect to see primer-dimers?
Melt curve detects 2 dsdnas
TaqMan probes for qpcr Forward and Reverse primers TaqMan probe: - Complementary to region of interest - fluorescent dye (R) quenched by Quencher (Q)
TaqMan qpcr 5 à3 nuclease activity of Taq polymerase Release of (R) à fluorescence, R accumulates during PCR
Fluorescence signal is specific Probe only fluoresces if it binds to target. Need three oligos to bind to get fluorescence; more specific than SYBR green detection Can prepare TaqMan probes w/different fluorophores different colors. Multiplex qpcr: measure amplification of multiple targets in one sample
SYBER Green Cheaper buy two probes Moderate specificity TaqMan probes Must purchase specific TaqMan probe Very specific Variable, ~100 copies Can detect 1-10 copies No multiplexing Can do multiplex qpcr
Components of qpcr
Hot start Taq polymerase Taq is inhibited at the start chemical modification (our enzyme) Antibody or small protein binding Inhibition is reversed, Taq is activated by heat Our enzyme requires 10 min. at 95 o C Misprimed sequences at low temp will not be extended. Set up reactions and store at 4 o C until ready to run.
Amount of template Less than PCR Too much template could result in <100% efficiency 10 1000 copies of target sequence gdna: 0.1 100 ng cdna from 0.001 1000 ng total RNA Wide range need to optimize
Primer Design Similar rules as for PCR. Product should be between 85 200 bp Want extension to be efficient Make sure the piece is in the cdna pool, long fragments may not be present
Contamination qpcr is very sensitive small amounts of contaminating template can interfere Use filter tips Take extra care