What is melting curve analysis in qPCR?

Melt curve analysis is frequently used as a diagnostic tool for assessing qPCR amplicon length with intercalating dye qPCR assays. Here, we explain how melt curves are produced, examine the assumptions being used, and describe some additional methods that can be used to further analyze melt curve results.

What is melting curve in real time PCR?

A melting curve charts the change in fluorescence observed when double-stranded DNA (dsDNA) with incorporated dye molecules dissociates, or “melts” into single-stranded DNA (ssDNA) as the temperature of the reaction is raised.

What are the steps of real time PCR?

Real-time PCR steps Figure 1 Real-time PCR involves conversion of RNA to cDNA via reverse transcription, followed by several rounds of PCR to amplify and detect the genes of interest. The products can be detected in ‘real-time’ by using SYBR-green or Taqman probes.

How do you analyze qPCR data?

There are two main ways to analyze qPCR data: double delta Ct analysis and the relative standard curve method (Pfaffl method). Both methods make assumptions and have their limitations, so the method you should use for your analysis will depend on your experimental design.

How do you analyze PCR results?

After a traditional PCR has been completed, the data are analyzed by resolution through an agarose gel or, more recently, through a capillary electrophoresis system. For some applications, a qPCR will be run with the end-point data used for analysis, such as for SNP genotyping.

What is the difference between PCR and real-time PCR?

RT–PCR is a variation of PCR, or polymerase chain reaction. The two techniques use the same process except that RT–PCR has an added step of reverse transcription of RNA to DNA, or RT, to allow for amplification.

What is the melt curve analysis?

The melt curve analysis is a protocol that is based on a standard PCR assay and incorporates fluorescent dye and a melting protocol following traditional PCR cycling. Distinguishing the different PCR products is based on the relative fluorescence intensity with amplicon melting.

When is data collection enabled in the melt curve protocol?

Data collection was enabled at the extension step. The melt curve protocol followed with 10 seconds at 95°C and then 10 seconds each at 0.2°C increments between 62°C and 95°C. Data collection was enabled at each increment of the melt curve.

What are the primers and results for a melt curve analysis?

While the primers section for a melt curve analysis will be the same as for a standard PCR, the results section will tell you what results to expect when you run this protocol as a melt curve analysis. This information is provided in the form of melting temperatures, or Tm.

What are the advantages of melting curve analysis for SNP detection?

The melting curve analysis can detect the presence of SNP/mutation and identify the type of nucleotide substitution. Each of the techniques does have its advantages and disadvantages, but one of the defining parameters is the downstream requirements.