BACKGROUND: High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49: 396-406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides.
METHODS: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), beta-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye
LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR.
RESULTS: The six common beta-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve
acquisition required only 1-2 min, amplification and analysis were achieved in 10-20 min with rapid cycling conditions. CONCLUSIONS: High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR
for amplicon genotyping, and is a promising method for mutation screening.

Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis.
Robinson BS, Monis PT, Dobson PJ.
Appl Environ Microbiol. 2006 Sep;72(9):5857-63.

Australian Water Quality Centre, Private Mail Bag 3, Salisbury, SA 5108, Australia

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at

close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and
investigation of the effect of dye concentration on amplification and DNA melting curve analysis.
Monis PT, Giglio S, Saint CP.
Anal Biochem. 2005 340(1): 24-34.
Microbiology Unit, Australian Water Quality Centre, Private Mail Bag 3, Salisbury, SA 5108, Australia.

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the

intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular amplicons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.

The use of new probes and stains for improved assessment of cell viability and
extracellular polymeric substances in Candida albicans biofilms.
Jin Y, Zhang T, Samaranayake YH, Fang HH, Yip HK, Samaranayake LP.
Mycopathologia. 2005 159(3): 353-60.
Division of Oral Biosciences, Faculty Dentistry, The Prince Philip Dental
Hospital, University of Hong Kong, 34 Hospital Road, SAR, China.

Phenotypic and genotypic cell differentiation is considered an important feature that confers enhanced antifungal resistance in candidal biofilms. Particular emphasis has been placed in this context on the viability of biofilm subpopulations, and their heterogeneity with regard to the production of extracellular polymeric substances (EPS). We therefore assessed the utility of two different labeled lectins Erythrina cristagalli (ECA) and Canavalia ensiformis (ConA), for EPS visualization. To evaluate the viability of candidal biofilms, we further studied combination stains, SYTO9 and propidium iodide (PI). The latter combination has been successfully used to assess bacterial, but not fungal, viability although PI alone has been previously used to stain nuclei in fungal cells. Candida albicans biofilms were developed in a rotating disc biofilm reactor and observed in situ using confocal scanning laser microscopy (CSLM). Our data indicate that SYTO9 and PI are reliable vital stains that may be used to investigate C. albicans biofilms. When used together with ConA, the lectin ECA optimized EPS visualization and revealed differential production of this material in mature candidal biofilms. The foregoing probes and stains and the methodology described should help better characterize C. albicans biofilms in terms of cell their viability, and EPS production.

EvaGreen® dye is a green fluorescent nucleic acid dye with features that make the dye useful for several applications including qPCR, high-resolution DNA melt curve analysis (HRM)1, real-time monitoring of thermophilic helicase-dependent amplification (tHDA), routine solution DNA quantification and capillary gel electrophoresis. The DNA-bound dye has excitation and emission spectra very close to those of fluorescein (FAM) or SYBR® dye Green I, making the dye readily compatible with instruments equipped with the 488 nm argon laser or any visible light excitation with wavelength in the region. EvaGreen dye is extremely stable both thermally and hydrolytically, providing convenience during routine handling. The dye is essentially nonfluorescent by itself, but becomes highly fluorescent upon binding to dsDNA. EvaGreen dye is nonmutagenic and noncytotoxic by being completely impermeable to cell membranes, unlike SYBR Green I, which enters cell rapidly and is known to be a powerful mutation-enhancer (Ohta, et el. Mutat. Res. 492, 91(2001).

The unique properties of EvaGreen dye have made it particularly useful in quantitative real-time PCR (qPCR) application. Compared with the widely used SYBR Green I, EvaGreen dye is generally less inhibitory toward PCR and less likely to cause nonspecific amplification. As a result, EvaGreen dye can be used at a much higher dye concentration than SYBR Green I, resulting in more robust PCR signal.

Summary of Mutagenic Toxicity Test Results for EvaGreen
Nucleic Acid Detection Technologies
Compiled by Biotium, Inc. from the results of an independent testing service:  Litron Laboratories, Inc., Rochester, NY

DNA quantification using EvaGreen and a real-time PCR instrument.
Weijie Wanga, Kunsong Chena and Changjie XuCorresponding Author Contact Information, a, E-mail The Corresponding Author
aState Agricultural Ministry Laboratory of Horticultural Plant Growth, Development and Biotechnology, Huajiachi Campus, Zhejiang University, Hangzhou 310029, People’s Republic of China
Analytical Biochemistry
Volume 356, Issue 2, 15 September 2006, Pages 303-305

DNA quantification is an important, frequently used technique, and inaccuracies can result in failures with ligation, restriction, polymerase chain reaction (PCR),1 amplified fragment length polymorphism (AFLP), Southern blotting, and other techniques. DNA is most commonly quantified using absorbance at 260 nm, but because of the existence of many impurities, this can be an imprecise measurement and DNA levels can be more than 10 times overestimated in some cases [1]. Quantification by agarose gel electrophoresis with a known amount of standard DNA [1] and [2] can provide more accurate data, but the procedures are complicated, the data often still are not accurate enough, and the technique is impractical for routine or high-throughput DNA quantification [3]. Fluorescence spectroscopy using various DNA intercalating dyes is the most widely accepted technique for accurate DNA quantification [4]. However, if the analysis is to be carried out with a fluorescence spectrophotometer, a relatively large assay volume (e.g., 2 ml) is required [5], and this is impractical for small DNA samples and expensive dyes. Fluorescence can also be measured with a smaller volume of DNA sample using other instruments such as fluorescent microplate readers [6], microplate fluorometers [7] and [8], and transilluminator–microplate–CCD camera systems [9], but the instruments might not be readily available in most molecular biology laboratories.

Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications.
Mao F, Leung WY, Xin X.
BMC Biotechnol. 2007 7(1): 76

Capillary electrophoresis of double-stranded DNA fragments using a new fluorescence intercalating dye EvaGreen.
Sang F, Ren J.
J Sep Sci. 2006 29(9): 1275-1280.
College of Chemistry and Chemical Engineering, Shanghai Jiaotong University, Shanghai, P. R. China.

HRM with SYBR Green (by Corbett Life Science)

So-called “new generation saturation dyes”, specifically LCGreen® and LCGreen Plus® (Idaho Technology), are promoted by some as essential for successful HRM analysis (Reed, Kent & Wittwer, 2007) although other dyes such as EvaGreen™ (Biotium Inc.) and SYTO®9 (Invitrogen) have been used with similar success (Krypuy et al 2006; Jeffery et al 2007; Wojdacz & Dobrovic 2007).

Moreover, the use of SYBR® Green 1 (SYBR) for HRM is actively discouraged by some authors (Wittwer et al 2003; Reed, Kent & Wittwer, 2007). Liew et al (2004) state that SYBR can only be used for HRM after substantial modification of the protocol (including the use of GC-clamps, triple primers, and allele-specific PCR). By contrast, we and others (Price et al 2007; Pornprasert et al 2008) have found SYBR to be a very successful dye for HRM analysis that does not require any protocol modifications. We unhesitatingly recommend its use with a Rotor-Gene 6000.

Prejudice against SYBR stems from early evidence collected on instrumentation that was not ideal for the task. Original assertions were based on experiments done with a pre-HRM era LightCycler™ capillary-based real-time analyzer (Roche Molecular Systems) using templates that included a low mass DNA size ladder (Wittwer et al 2003; Liew et al 2004). The reason SYBR was less successful than LCGreen was “not entirely clear” at the time, but a “dye redistribution” hypothesis was suggested (Wittwer et al 2003). According to this hypothesis, SYBR dye releases from low-temperature duplexes during melting and dynamically re-intercalates into neighboring duplexes that melt at higher temperature. This mechanism became the basis for the “dye saturation model” (Wittwer et al 2003, Liew et al 2004). According to this model, if sufficiently high concentrations of dye are used such that all binding positions on the DNA are occupied (i.e. saturated), then dye redistribution effects are minimized and greater melting curve resolution can be achieved. So-called “saturating dyes” were defined as those that can be used at concentrations sufficiently high to saturate all DNA binding sites without inhibiting the PCR. This model formed the basis for IP protection and a patent by Witter et al and Idaho Technology.

In spite of the saturation model, mounting evidence clearly shows that it is not valid on the Rotor-Gene 6000 HRM instrument. This is true for a range of dyes, including SYBR (manuscript in preparation). In fact, SYBR used at standard non-saturating concentrations is highly suitable for HRM analysis, as illustrated below for the detection of a Class 4 SNP. We therefore urge users not to dismiss the use of SYBR for HRM.

SensiMix HRM     (Quantace) SensiMix HRM has been designed for High Resolution Melt (HRM) analysis on the Rotor-Gene 6000. To learn more about the applications of HRM and to see some data from our mix, please click here.
FEATURES     HRM optimised mix with a separate vial of EvaGreen dye.     Ultra-high sensitivity: detects class 4 (A/T) SNP mutations.     Comes in 250, 500 and 2000 (25µl) reaction packs.     HRM™ demo kit containing this mix is available to Corbett Life Science Distributors.

TATAA Biocenter, Odinsgatan 28, 41103 Goteborg, Sweden

Chromofy is a monomeric asymmetric cyanine dye, developed by TATAA Biocenter for use in qPCR applications. The dye has absorbance and emission wavelengths that are suitable for the FAM/SYBR channel on most common real-time PCR instruments. When binding to dsDNA Chromofy shows a very strong fluorescence increase.

High-resolution melting is a post-PCR analysis that discriminates different samples on the basis of their melting temperature. Chromofy and HRM can be used for genotyping, either with ro without unlabelled probes and for methylation analysis. Methylation analysis is performed after bisulfite treatment of the DNA samples which converts unmethylated cytosines to uracils while methylated cytosines remain intact. During subsequent PCR the uracils will be substituted for thymines giving rise to a Tm difference between methylated and un-methylated samples, which is seen as varying heights of the plateu in the normalized HRM data. Using Chromofy your assay can be sensitive down to 1 % methylated DNA in un-methylated background.

Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis
Kristina Lind1,2, Anders Ståhlberg2,3, Neven Zoric2, and Mikael Kubista1,2
1 Chalmers University of Technology, Gothenburg, 2 TATAA Biocenter, Gothenburg, and 3 Lund University, Lund, Sweden
BioTechniques 40:315-319 (March 2006)

Currently, in real-time PCR, one often has to choose between using a sequence-specific probe and a nonspecific double-stranded DNA (dsDNA) binding dye for the detection of amplified DNA products. The sequence-specific probe has the advantage that it only detects the tar-geted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplification, which reveals what kind of products have been formed. Here we present a new strategy based on combining a sequence-specific probe and a nonspecific dye, BOXTO, in the same reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan® probes and locked nucleic acid (LNA) probes without interfering with the PCR. The probe signal reflect formation of target product, while melting curve analysis of the BOXTO signal reveals primer-dimer formation and the presence of any other anomalous products.

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BEBO is an unsymmetric cyanine dye developed by TATAA Biocenter for use in qPCR applications.

A new minor groove binding asymmetric cyanine reporter dye for real-time PCR
Martin Bengtsson, H. Jonas Karlsson, Gunnar Westman and Mikael Kubista*
Department of Chemistry and Bioscience, Chalmers University of Technology 41296 Goteborg and
TATAA Biocenter, Odinsgatan 28, 41103 Goteborg, Sweden
Nucleic Acids Research, 2003, Vol. 31, No. 8 e45

The minor groove binding asymmetric cyanine dye 4-[(3-methyl-6- (benzothiazol-2-yl)- 2,3-dihydro- (benzo-1,3-thiazole) -2-methylidene)]- 1-methyl-pyridinium iodide (BEBO) is tested as sequence nonspeciÆc label in real-time PCR. The Fluorescence intensity of BEBO increases upon binding to double-stranded DNA allowing emission to be measured at the end of the elongation phase in the PCR cycle. BEBO concentrations between 0.1 and 0.4 mM generated sufÆcient Øuorescence signal

Pre-validated HRM assays    (by Primer Design)