Fluorescence resonance energy transfer (FRET) is a simple procedure for detecting specific DNA sequences, and is therefore used in many fields. However, the cost is relatively high, because FRET-based methods usually require fluorescent probes. We have designed a cost-effective way of using FRET, and developed a novel approach for the genotyping of single nucleotide polymorphisms (SNPs) and all...

Using fluorescence microscopy, we explored the ability of cultured immune cells to take up aqueous SYBR Green I (SGI). SYBR Green I, a highly sensitive fluorescent nucleic acid stain, which preferentially binds to dsDNA over ssDNA or RNA with little background fluorescence from unbound molecules. A time course study over 2h using final dilutions of SGI of 1:10,000 and 1:100,000 at 22 and 37 deg...

We have evaluated a real-time PCR procedure based on the LightCycler technology for rapid detection of herpes simplex virus (HSV) in genital lesions. Two sets of primers, corresponding to the thymidine kinase and DNA polymerase regions, were used for the amplification reactions in separate capillaries containing the SYBR Green I dye as detection signal. In 28 of 118 samples (24%), HSV was isola...

The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical applications over the last decade. Many applications utilize SYBR Green I dye to follow the accumulation of amplicons in real time. SYBR Green I has, however, a number of limitations that include the inhibition of PCR, preferential binding to GC-rich sequences and effects on melting curve analysis. Al...

Real-time reverse transcription polymerase chain reaction (RT-PCR) methods that monitor product accumulation were adapted for the validation of differentially expressed genes. We describe a real-time quantitative PCR assay that uses SYBR Green I dye-based detection and product melting curve analysis to validate differentially expressed genes identified by gene expression profiling technologies....

Standardized procedures must be followed when characterizing, officially describing, and validly naming novel bacteria. For species descriptions, DNA-DNA hybridization still is needed for whole-genome comparisons between close relatives, but many established hybridization methods have drawbacks, such as requiring labeled or large amounts of DNA. We evaluated a new technique based on the spectro...

BACKGROUND DNA melting analysis for genotyping and mutation scanning of PCR products by use of high-resolution instruments with special "saturation" dyes has recently been reported. The comparative performance of other instruments and dyes has not been evaluated. METHODS A 110-bp fragment of the beta-globin gene including the sickle cell anemia locus (A17T) was amplified by PCR in the presenc...

PE Biosystems has developed guidelines enabling streamlined design and implementation of real-time quantitative PCR assays. The use of these guidelines makes it easy to apply either the fluorogenic 5 ́ nuclease assay or SYBR Green I double-stranded DNA binding dye chemistry to any real-time quantitative PCR system. Specific assay design and optimization guidelines minimize the time and cost of a...

PE Biosystems has developed guidelines enabling streamlined design and implementation of real-time quantitative PCR assays. The use of these guidelines makes it easy to apply either the fluorogenic 5 ́ nuclease assay or SYBR Green I double-stranded DNA binding dye chemistry to any real-time quantitative PCR system. Specific assay design and optimization guidelines minimize the time and cost of a...

SYBR Gold staining is used for enumerating bacteria and viruses in aquatic samples. However, its suitability for epifluorescence microscopy has not been sufficiently investigated. Thus we compared bacterial and viral counts using SYBR Gold and SYBR Green I stains. Variables for both bacterial and viral counts included season and ocean depths of sample collection and the period of sustained exci...