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Detection of RB Germline Mutations Using Exon-by-exon heteroduplex Analysis Compared with SSCP

Eye Science 1996年第3期16卷 151-157页

作者： Kensei Minoda Department of Ophthalmology Teikyo University Ichihara Hospital

Purpose: To compare heteroduplex analysis with SSCP and to develop a simple and effective method for mutational screening of RB *** and Methods: Leukocyte DNA was prepared from 12 unrelated Japanese patients with hereditary retinoblstoma. PCR combined with simultaneous nonisotopic heteroduplex and SSCP analysis was used to screen leukocyte DNA for such mutations, exon-by-exon, without the use of restriction endonu-clease digestion. PCR was conducted using 28 pairs of primers flanking all 27 ex-ons and the promoter region of the RB gene, with PCR products ranging from 159bp to 326bp. Mutations were identified by ***: Heterozygous germline mutations were detected in 8 of 12 Japanese patients. The mutations were identified by sequencing as follows; G→C/acceptor of exon 11, T insertion/codon 389, C→T/codon 455, 33bp insertion/codon 455 (C GA), G→T/codon 533, C→T/codon 579, C deletion/codon 674, and C→T/ codon ***: Our results suggest that small RB gene mutations

关键词： retinoblastoma gene mutation SSCP heteroduplex

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Photophysical Behavior of heteroduplexes Formed from Naphthalene Incorporated Foldamers,and Linear Donors and Pyromellitic Diimide Incorporated Foldamers

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Chemical Research in Chinese Universities 2009年第3期25卷 317-327页

作者： ZHAO Shu-min GU Hai-ning ZHOU Qi-zhong Key Laboratory for Molecular Design and Nutrition Engineering of Ningbo Ningbo Institute of Technology of Zhejiang University Ningbo 315100 P. R. China Department of Chemistry Zhejiang University Hangzhou 310027 P. R. China

The authors synthesized the electron-rich 1,5-dioxonaphthyl group incorporated foldamers, and linear donors and electron-deficient pyromellitic diimide incorporated foldamers, which were investigated in organic solvents. There is an evident charge transfer process from the donors to the electron-deficient foldamers, due to the donor-acceptor interaction of the heteroduplexes formed from the donors and the electron-deficient foldamers in chloroform, observed by using the ultraviolet（UV） visible method. The charge transfer process of the heteroduplexes depends on the molecular structure（not only the number of 1,5-dioxonaphthyl groups and pyromellitic diimide groups in the heteroduplex, but also the intermolecular double hydrogen bond in the heteroduplex）, the intercalative π-π stacking motif, the initial concentration of heteroduplexes, and the polarity of the solvent. Fortunately, the authors found a critical bumping concentration（CBC） of the donors in acetonitrile or chloroform, which is useful in quantitative research, by using the fluorescence method. The value of CBC depends not only on intramolecular folding and the intramolecular hydrogen bond, but also on intramolecular π-π stacking and the solvent. The electron-deficient foldamers quenched the fluorescence of the donors. Fluorescence quenching depends not only on the number of pyromellitic diimide groups and 1,5-dioxonaphthyl groups in the heteroduplex, but also on the intercalative π-π stacking motif between the donor and the acceptor.

关键词： heteroduplex Foldarner Linear donor Charge transfer Quench

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A Simple LDT-PCR Method for Detection of Various Nucleic Acid Sequence Changes in a Small Region of Genes—Application for the Identification of Gene Mutations in MTB <i>rpoB</i>Gene Associated with Drug Resistance

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Open Journal of Medical Microbiology 2013年第3期3卷 172-177页

作者： Xing Tang Steven Wood Larissa Baeva Division of Biology OSEL/CDRH/FDA

A modified low denaturing temperature PCR (LDT-PCR) method combined with DNA microarray technique is developed in our lab for quick and effective identification of various mutations in an 81 base pair region of Mycobacterium Tuberculosis (MTB) ribosome RNA polymerase subunit B (rpoB) gene associated with rifampin resistance. By incurporation of wild type (wt) allele fragments that had been PCR amplified previously, the target PCR fragments coming from mutant clinical MTB samples were codenaturized with incorporated wt type allele fragment at 94°C and then let them randomly form matched structures (homoduplex) and allele mismatch-containing structures (heteroduplex), respectively, when the temperature cooled down to 70°C. After the temperature was raised to 80°C, the heteroduplex double stranded fragments were preferentially denatured and resulted in PCR amplification as well as fluorescence incurporation. Since the homoduplex fragments need a higher temperature to be denatured, they were kept in double-stranded status at that temperature and failed to be PCR amplified. By hybridization of LDT-PCR products with the probes spotted on microarray slides, the fluorescent signals representing the presence of gene mutations were detected. We have tested this method on 35 clinical MTB samples and obtained satisfied results.

关键词： Low Denaturing Temperature PCR (LDT-PCR) Mycobacterium Tuberculosis (MTB) Ribosome RNA Polymerase Subunit B (rpoB) Drug Resistance Homoduplex heteroduplex Microarray

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