Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

BACKGROUND: Melting temperature analysis of products amplified with SYBR I Green chemistry is a cheap and effective method for identification of sequence differences. When used in conventional quantitative real-time PCR instruments (qPCR), this method is limited by temperature variations over the heating block and low numbers of fluorescence measurements during the dissociation step, which hamper the ability of most instruments to report accurate and precise melting temperatures. METHODS: We designed a molecular beacon-based temperature indicator probe (Tm-probe) to control for variations in temperatures over the heating block of the instrument. In addition, we wrote an automated curve-fit analysis algorithm of dissociation data to use multiple data points with a gaussian curve fit to extrapolate precise melting temperatures. RESULTS: Use of the Tm-probe in conjunction with the analysis algorithm and multiple dissociations improved SDs of melting temperatures over a 96-well plate from 0.19 to 0.06 degrees C. CONCLUSIONS: Melting temperature analyses with SYBR I Green chemistry on conventional qPCR instruments can be improved by the use of a Tm-probe in conjunction with curve-fit analysis of data. Resolution improvement up to 3-fold is possible and allows additional melting temperatures to be identified.

Original publication




Journal article


Clinical chemistry

Publication Date





98 - 103


Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.


Organic Chemicals, Rhodamines, Oligonucleotide Probes, Fluorescent Dyes, Autoanalysis, Polymerase Chain Reaction, Sequence Analysis, Algorithms, Transition Temperature