How Real Time qPCR Works

Real-time PCR, also known as quantitative or qPCR, determines the actual amount of PCR product present at a given cycle. By using a fluorescent report in the PCR reaction, this process allows you to measure DNA generation in the qPCR assay. There are two methods of qPCR: SYBR Green and probe-based.

SYBR Green-based qPCR

SYBR Green qPCR allows monitoring of amplification of any double-stranded DNA sequence. It does not require probes and thus reduces assay setup and running costs. Also, multiple dyes can bind to a single amplified molecule, which increases sensitivity for detecting amplification products.

SYBR Green qPCR Video Transcript

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Real-time PCR, also known as quantitative or qPCR, determines the actual amount of PCR product present at a given cycle. By using a fluorescence report in the PCR reaction, this process allows you to measure DNA generation in the qPCR assay. 

In a SYBR Green qPCR reaction you have your template, which contains the target sequence that you are interested in. You also need primers, dNTPs and DNA polymerase of your choosing. The SYBR Green I dye is typically included in the reaction mix that contains the DNA polymerase.

Denaturation is the first step in the PCR reaction. The thermocycler heats up to roughly 95 degrees Celsius, which causes the double-stranded DNA helix to melt open into two single-stranded DNA templates.

During the annealing step, the temperature cools between 45-65 degrees Celsius and the single-stranded primers attach to the appropriate ends of the target sequence. During the cycle, DNA polymerase attaches to the primed template and begins to incorporate complementary nucleotides.

Finally, during extension, the temperature slightly rises to 65-75 degrees Celsius. DNA polymerase extends the sequence-specific primer with the incorporation of nucleotides that are complementary to the DNA template.

SYBR Green I binds all newly synthesized double-stranded DNA complexes and fluoresces. The fluorescence accumulates as cycling of PCR continues and is measured at the end of each PCR cycle. The intensity of fluorescence generated by SYBR Green I above background level (the Cq value) is measured and used to quantitate the amount of newly generated double-stranded DNA.

After repeating the denaturation, annealing and extension cycles approximately 35-40 times, you are ready to begin analysis. The Cq values can be used to quantitate starting amounts of DNA, establish a standard curve for gene expression studies or other analysis.

SYBR Green qPCR is a great option for monitoring amplification of any double-stranded DNA sequence. It does not require probes and thus reduces assay setup and running costs. Also, multiple dyes can bind to a single amplified molecule, which increases sensitivity for detecting amplification products.

Instrument compatibility is critical for qPCR. Be sure to select the right product for your instrument. In addition, qPCR methods vary on levels of fidelity, speed and convenience. We recommend using a PCR selection guide and our technical support team to select what products are right for you at SigmaAldrich.com/PCR.

Probe-based qPCR

Probe-based qPCR allows for specific hybridization. The targeted nature of probe-based qPCR leads to low background and eliminates the presence of false positives. You can also label probes with different, distinguishable reporter dyes to allow for amplification of two distinct sequences in one reaction tube.

Probe-based qPCR Video Transcript

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Real-time PCR, also known as quantitative or qPCR, determines the actual amount of PCR product present at a given cycle. By using a fluorescence report in the PCR reaction, this process allows you to measure DNA generation in the qPCR assay. 

In a probe-based qPCR reaction you have your template, which contains the target sequence that you are interested in. You also need primers, dNTPs and DNA polymerase of your choosing. In addition, you will need a probe labeled with both a reporter molecule and quencher molecule. Typically probes are sold as separate, custom products because they are specific to the target sequence.

Denaturation is the first step in the PCR reaction. The thermocycler heats up to roughly 95 degrees Celsius, which causes the double-stranded DNA helix to melt open into two single-stranded DNA templates.

During the annealing step, the temperature cools between 45-65 degrees Celsius and the single-stranded primers attach to the appropriate ends of the target sequence. During the cycle, DNA polymerase attaches to the primed template and begins to incorporate complementary nucleotides.

Finally, during extension, the temperature slightly rises to 65-75 degrees Celsius. During this phase, DNA polymerase extends the sequence-specific primer with the incorporation of nucleotides that are complementary to the DNA template. The DNA polymerase then displaces the reporter molecule from the probe resulting in fluorescence.

The fluorescence accumulates as cycling of PCR continues and is measured at the end of each PCR cycle. The intensity of the fluorescence generated by the reporter molecule above background level (the Cq value) is measured and used to quantitate the amount of newly generated double-stranded DNA strands.

After repeating the denaturation, annealing and extension cycles approximately 35-40 times, you are ready to begin analysis. The Cq values can be used to quantitate starting amounts of DNA, establish a standard curve for gene expression studies or other analysis.

Probe-based qPCR is a great option for specific hybridization, no false positives, and amplification of two distinct sequences in one reaction tube.

Learn more about our complete PCR portfolio and how to select the right reagents for your experiment.