Real-Time Quantitative PCRBasis

ABI Prism 7900HT real-time PCR instrument

Content Principles of quantificationMethods of quantificationApplications

The polymerase chain reaction (PCR) has revolutionized the detection of DNA and RNA. As little as a single copy of a particular sequence can be specifically amplified and detected. Theoretically, there is a quantitative relationship between amount of starting target sequence and amount of PCR product at any given cycle. In practice, though, it is a common experience for replicate reactions to yield different amounts of PCR product. The development of real-time quantitative PCR has eliminated the variability traditionally associated with quantitative PCR, thus allowing the routine and reliable quantitation of PCR products.

PCR Reaction Phase

Quantification in Log PhaseInitial template ,more significantPerfect reproducibility, less errorConstant amplification efficiency, linear standard curve

Several Basic Concepts Threshold: The low limit when PCR reaction signal goes into log phaseCt value: The cycle number of PCR reaction when the fluorescence signal reaches to threshold

Standard CurveCT = -k lgX0+ bThere is a linear relationship between CT and lgX0 (X0 stands for amount of initial DNA)Use standard sample which we have known its initial concentration to make standard curve. And then we can identify the initial concentration of unknown template through the standard curve when we obtain its CT value.

Methods of quantificationDye method: SYBR Green IProbe methods: TaqMan Probe and TaqMan MGB ProbeMolecular beaconsDual-oligo FRET pairs

SYBR Green I SYBR Green I dye is a highly specific double-stranded DNA binding dye. Its fluorescence increases when it bound to double-stranded DNA while disappears when DNA denatured.There is a direct ratio between fluorescence signal and the amount of double-stranded DNA.

Advantages and Disadvantages about SYBR Green I SYBR Green I assay chemistry will detect all double-stranded DNA, including non-specific reaction products.The advantage of SYBR Green I assay chemistry is that no probe is required, thus reducing assay setup and running costs. And it can make dissociation curve analysis.

Fluorescent Resonant Energy Transfer (FRET)When the emission band of one fluorophore is overlapped with the absorption band of another, and simultaneously they are very near , then the energy will transfer from short wavelength (high energy) fluorophore to the longer wavelength (lower energy ) one. In other words, the fluorescence of short wavelength fluorophore is quenched by another.

TaqMan Probe One probe, two primers.One probe, two fluorophores: one is reporter, another is quencher .Hybridises with the target ampliconIs 3 terminally blocked (cannot be extended by the polymerase)Has two fluorescent dyes attached: 1.Reporter(R) 2.Quencher(Q)

React Process

The production of one DNA strand will cut one probe into pieces simultaneously.The disjunction of one probe will produce one fluorescent signal.Signal intensity are proportional to the amount of DNA which is binded by probe.

Advantages about TaqMan chemistryNoiseless data Due to the second level of specificity provided by the probe.Multiplex compatible Each probe can be differently colored and thereby mixed with others.Signal proportional to products Signal related to amount of amplified product .Unreversible reaction, signal will not quenched Other probe chemistries use reversible hybridisation to generate signal.

TaqMan MGB ProbeNon background fluorescenceAll probes will be short(13-20bp) Tm enhancer: MGBMinor Groove Binder attached at 3' end of probe

SNP Discrimination (Allele Discrimination )

ResultHomozygote: wild type: FAM mutant type: VICHeterozygote: FAM+VIC

Other Methods Based on Probe

Molecular beacons

Dual-oligo FRET pairs

Applications: Primer and Probe Design Using Primer Express SoftwarePrimer Express software uses a set of default parameters to automatically select primer and probe sets. Even though no probe is required for SYBR Green I dye detection, it is still a good idea to use Primer Express software to select a primer and probe set when designing a SYBR Green I assay. Although no probe will be used, the primers will meet all the required criteria and if, in the future, there is the need to convert the assay to TaqMan assay chemistry to obtain higher specificity, the probe can immediately be found in the original Primer Express software document.

Primer and Probes selection guidelines for quantitative assays

ApplicationsAbsolute Quantification (AQ)Absolute--produces data with quantity amountsThe absolute quantity of target gene is measuredneeds a standard template whose concentration is known absolutelyThe quantity of the standard template has to be measured preciselyunnecessary for most studies

ApplicationsRelative Quantification (RQ)Relative--makes comparisons of quantity (no units) relative standard curve or CT analysisany stock DNA or RNA containing the target gene can be used to prepare relative standard curvenormalises for amount of sample addedneeds endogenous control targetthe most powerful and widely used method

ApplicationsAllelic Discrimination (AD)Plus/Minus with IPC (+/-)