Monthly Archives: April 2016
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- 四月 27, 2016
In this next installment on how to improve your qPCR gene profiling (see 6 Tips to Improve qPCR for previous discussion), we would like to discuss the limitations of melting curve analysis and PCR inhibitors.
Tip #3. Melting curve analysis is NOT sufficient for assessing qPCR product specificity.
In a melting curve analysis, the number of peaks is commonly used to determine the purity of qPCR products, but is this accurate enough? While having only one peak may be a good indicator of product purity, melting curve analysis can produce both false negatives and positives. Here’s how:
In dye-based qPCR, a DNA-binding dye (e.g., SYBR® Green) fluoresces when bound to double-stranded DNA (dsDNA, i.e. your qPCR product), and this increase in fluorescence as dsDNA is produced over time is quantified. In contrast, a melting curve analysis is essentially the opposite measurement. In a melting curve analysis, temperature is gradually increased to "melt" or dissociate dsDNA products. As
