title: Molecular beacon-based temperature control and automated analyses for improved resolution of melting temperature analysis using SYBR I green chemistry.
abstract: 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.
authors.affiliation: Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. christoffer.nellaker@ki.se
authors.forename: Christoffer
authors.initials: C
authors.lastname: Nellåker
authors.forename: Ulf
authors.initials: U
authors.lastname: Wållgren
authors.forename: Håkan
authors.initials: H
authors.lastname: Karlsson
mesh_terms: D000465:Algorithms; D001322:Autoanalysis; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes / Q000737:chemistry*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D009930:Organic Chemicals / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012235:Rhodamines / Q000737:chemistry; D017421:Sequence Analysis / Q000379:methods*; D044366:Transition Temperature
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D011804:Quinolines; D012235:Rhodamines; C098022:SYBR Green I