References >> qPCRReal Time PCR
As the name suggests, real time PCR is a technique used to monitor the progress of a PCR reaction in real time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified. Real Time PCR is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds. This occurs due to the accumulation of the PCR product with each cycle of amplification. These fluorescent reporter molecules include dyes that bind to the double-stranded DNA (i.e. SYBR® Green ) or sequence specific probes (i.e. Molecular Beacons or TaqMan® Probes). Real time PCR facilitates the monitoring of the reaction as it progresses. One can start with minimal amounts of nucleic acid and quantify the end product accurately. Moreover, there is no need for the post PCR processing which saves the resources and the time. These advantages of the fluorescence based real time PCR technique have completely revolutionized the approach to PCR-based quantification of DNA and RNA. Real time PCR assays are now easy to perform, have high sensitivity, more specificity, and provide scope for automation. Real time PCR is also referred to as real time RT PCR which has the additional cycle of reverse transcription that leads to formation of a DNA molecule from a RNA molecule. This is done because RNA is less stable as compared to DNA
In a real time PCR protocol, a fluorescent reporter molecule is used to monitor the PCR as it progresses. The fluorescence emitted by the reporter molecule manifolds as the PCR product accumulates with each cycle of amplification. Based on the molecule used for the detection, the real time PCR techniques can be categorically placed under two heads:
1. Non-specific Detection using DNA Binding Dyes
2. Specific Detection Target Specific Probes
Non-specific Detection using DNA Binding Dyes
In real time PCR, DNA binding dyes are used as fluorescent reporters to monitor the real time PCR reaction. The fluorescence of the reporter dye increases as the product accumulates with each successive cycle of amplification. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase. If a graph is drawn between the log of the starting amount of template and the corresponding increase the fluorescence of the reporter dye fluorescence during real time PCR, a linear relationship is observed.
SYBR® Green is the most widely used double-strand DNA-specific dye reported for real time PCR. SYBR® Green binds to the minor groove of the DNA double helix. In the solution , the unbound dye exhibits very little fluorescence. This fluorescence is substantially enhanced when the dye is bound to double stranded DNA. SYBR® Green remains stable under PCR conditions and the optical filter of the thermocycler can be affixed to harmonize the excitation and emission wavelengths. Ethidium bromide can also be used for detection but its carcinogenic nature renders its use restrictive.
Although these double-stranded DNA-binding dyes provide the simplest and cheapest option for real time PCR, the principal drawback to intercalation based detection of PCR product accumulation is that both specific and nonspecific products generate signal.
Specific Detection using Target Specific Probes
Specific detection of real time PCR is done with some oligonucleotide probes labeled with both a reporter fluorescent dye and a quencher dye. Probes based on different chemistries are available for real time detection, these include:
1 . Quantitative mRNA expression studies.
2 . DNA copy number measurements in genomic or viral DNAs.
3 . Allelic discrimination assays or SNP genotyping.
4 . Verification of microarray results.
5 . Drug therapy efficacy.
6 . DNA damage measurement.
Real time PCR allows for the detection of PCR product during the early phases of the reaction. This ability of measuring the reaction kinetics in the early phases of PCR provide a distinct advantage over traditional PCR detection. Traditional methods use gel electrophoresis for the detection of PCR amplification in the final phase or at end-point of the PCR reaction.
In a PCR reaction as the reaction progresses, the reagents are being consumed as a result of amplification. Now the PCR product is no longer being doubled at each cycle due to this reagent constraint. This depletion will occur at different rates for each replicate. Thus, the samples begin to diverge in their quantities. This diminished amplification is the linear phase of the reaction. The plateau for each tube will differ due to the different reaction kinetics for each sample. It is in this phase where traditional PCR takes its measurement, also known as the end-point. This End-Point Detection has some problems such as low resolution, poor precision, low sensitivity and the need for post PCR processing. Also, the results of this detection are not expressed in numbers and there is no scope for automation.
Real Time PCR Primer and Probe Design with AlleleID® & Beacon Designer™
Beacon Designer™ is a comprehensive real time PCR primer and probe design tool for designing single template and multiplex assays. Real time PCR chemistries supported include Molecular beacons, TaqMan®, FRET, Scorpions® and SYBR® Green. It designs molecular beacons for standard and NASBA® assays and designs LNA spiked Taqman® probes as well. AlleleID® supports all the major features of Beacon Designer™ except NASBA® assay, LNA™ spiked Taqman® probe design and multiplex assay design. TaqMan® MGB probe design is unique to AlleleID® (whereas regular TaqMan® probes based assays can be designed using both AlleleID® and Beacon Designer™. AlleleID® is written for advance real time PCR applications such as oligo design for species identification and taxa discrimination. It includes a multiple sequence alignment module for identifying conserved and species specific regions. AlleleID® also includes support for real time PCR primer design over exon junctions for selective amplification of cDNA.