Assay of DNA Denaturation by Polymerase Chain Reaction-Driven Fluorescent Label Incorporation and Fluorescence Resonance Energy Transfer

Hiyoshi, M; Hosoi, Shigeru

doi:10.1006/abio.1994.1417

Cited by 22 publications

(8 citation statements)

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“…The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. Fluorescent chemistries commonly used include nonspecific compounds such as the DNA intercalating dyes SYBR® green (Schneeberger et al, 1995) and Syto9 (Monis et al, 2005) or sequence-specific oligoprobes that carry a donor and acceptor fluorophor and employ Fluorescent Resonance Energy Transfer (FRET) (Hiyoshi and Hosoi, 1994). While some product confirmation can be achieved with non-specific fluorescent dyes by performing denaturation (melting) curve analysis at the completion of the amplification, in practice, our experience has been that the specific approach yields better results for the detection of infectious agents in clinical specimens, and particularly so in the determination of pathogen load.…”

Section: Real-time Pcr Technologymentioning

confidence: 99%

Molecular Diagnosis of Medical Viruses

2007

Current Issues in Molecular Biology

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The diagnosis of infectious diseases has been revolutionized by the development of molecular techniques, foremost with the applications of the polymerase chain reaction (PCR). The achievable high sensitivity and ease with which the method can be used to detect any known genetic sequence have led to its wide application in the life sciences. More recently, real-time PCR assays have provided additional major contributions, with the inclusion of an additional fluorescent probe detection system resulting in an increase in sensitivity over conventional PCR, the ability to confirm the amplification product and to quantitate the target concentration. Further, nucleotide sequence analysis of the amplification products has facilitated epidemiological studies of infectious disease outbreaks, and the monitoring of treatment outcomes for infections, in particular with viruses which mutate at high frequency. This review discusses the applications of qualitative and quantitative real-time PCR, nested PCR, multiplex PCR, nucleotide sequence analysis of amplified products and quality assurance with nucleic acid testing (NAT) in diagnostic laboratories.

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Section: Real-time Pcr Technologymentioning

confidence: 99%

Molecular Diagnosis of Medical Viruses

2007

Current Issues in Molecular Biology

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“…FRET measurements have been widely used to detennine proximity relationship in biological macromolecules (23) (see also (24) for review) and nucleic acids structures (25)(26)(27). This approach has been recently applied to both duplex (27)(28)(29)(30)(31)(32)(33) and branched (34)(35)(36)(37) nucleic acid species labeled at the 5' tennini with dyes. Studies of the triplex formation have also been reported (38,39).…”

Section: Introductionmentioning

confidence: 99%

“…The rate of energy transfer depends on the spectral properties of the donor and acceptor, the relative orientation of their transition dipoles, and the distance between the donor and the acceptor chromophores (22). FRET measurements have been widely used to detennine proximity relationship in biological macromolecules (23) (see also (24) for review) and nucleic acids structures (25)(26)(27). This approach has been recently applied to both duplex (27)(28)(29)(30)(31)(32)(33) and branched (34)(35)(36)(37) nucleic acid species labeled at the 5' tennini with dyes.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Resonance Energy Transfer Analysis of the Degradation of an Oligonucleotide Protected by a Very Stable Hairpin

Réfrégiers¹,

Laigle²,

Jollès³

et al. 1996

Journal of Biomolecular Structure and Dynamics

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In vitro degradation of antisense oligonucleotides protected or not on their 3' side against enzymatic attack by a naturally forming hairpin has been studied by fluorescence resonance energy transfer (FRET). The two oligonucleotides d(5"TTCTCGCGAAGC3') forming the hairpin and d(5"TTCTCCGGAAGC3') as a control were labeled on their 5' side by tetramethylrhodamine and on their 3' side by fluorescein. Fluorescein has been shown not to hinder the hairpin formation and to give an additional protection against nucleases. The FRET technique proved adequate for an in situ study of these protected antisense oligonucleotides in living cells.

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“…Fluorescence-monitoring systems for qPCR consist of the following: 1) hydrolysis probes; 2) hybridizing probes; and 3) DNA-binding agents. The TaqMan assay is a typical system using the hydrolysis probe, for which fluorescence resonance energy transfer (FRET) technology is applied (Hiyoshi and Hosoi, 1994;Chen et al, 1997). The probe contains two labels, a fluorescent reporter dye at the 5 -end and a fluorescent quencher at the 3 -end.…”

Section: Kb the First Nucleotide (G/a) In Intron 17mentioning

confidence: 99%

Methods to detect and analyze copy number variations at the genome-wide and locus-specific levels

Lee

Jeon

2008

Cytogenet Genome Res

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Copy number variations (CNVs) have effects on phenotypes by altering transcription levels of genes and may have major impacts on protein sequence, structure and function. Therefore, CNV screening and analysis focused on the identification of CNV-genetic disease relations are actively progressing. CNVs can be detected and analyzed by various methodologies at the genome-wide and locus-specific levels. The genome-wide analysis of CNVs has been enhanced by bioinformatic tools for long-range sequence analysis, and comparative genome hybridization using microarrays containing either single nucleotide polymorphisms or bacterial artificial chromosome clones that represent the whole genome. RFLP followed by Southern blot analysis, quantitative real-time PCR, pyrosequencing, ligation detection reaction and the invader assay have become the main tools for locus-specific analysis so far. In this review, we present a brief principle, application history, and strengths and weaknesses of the methods used to detect CNVs at the genome-wide and locus-specific levels.

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Assay of DNA Denaturation by Polymerase Chain Reaction-Driven Fluorescent Label Incorporation and Fluorescence Resonance Energy Transfer

Cited by 22 publications

References 0 publications

Molecular Diagnosis of Medical Viruses

Molecular Diagnosis of Medical Viruses

Fluorescence Resonance Energy Transfer Analysis of the Degradation of an Oligonucleotide Protected by a Very Stable Hairpin

Methods to detect and analyze copy number variations at the genome-wide and locus-specific levels

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