caa a22 4500 397573987 CHVBK 20180308164849.0 cr unu---uuuuu 161202e199606 xx s 000 0 eng 10.1093/hmg/5.6.755 doi (NATIONALLICENCE)oxford-10.1093/hmg/5.6.755 Mutational Scanning of Large Genes By Extensive Pcr Multiplexing and Two-Dimensional Electrophoresis: Application to the RB1 Gene [Elektronische Daten] [Nathalie J. Van Orsouw, Daizong Li, Pieter van der Vlies, Hans Scheffer, Charis Eng, Charles H. C. M. Buys, Frederick P. Li, Jan Vijg] With the rapid increase in the number of identified human disease genes, the development of accurate and cost-efficient mutation tests has become opportune. Here we present a combination of extensive PCR multiplexing and two-dimensional (2-D) DNA electrophoresis to screen for mutations in 26 exons of the retinoblastoma (RB1) tumor suppressor gene. In 2-D electrophoresis, fragments are separated according to size and base pair sequence in non-denaturing and denaturing gradient gels, respectively. All target fragments, designed to have optimal melting characteristics, were prepared in a two-step PCR (a 6-plex long-PCR pre-amplification and a subsequent 25-plex short-PCR) followed by heteroduplexing. The mixture of PCR amplicons was then subjected to 2-D electrophoresis under a single set of experimental conditions. With this design, 35 previously identified mutations in 18 different exons were detected in 33 bilateral retinoblastoma patients. These results suggest that 2-D electrophoresis in this format provides a generally applicable, practical and fast way to diagnose with high accuracy large genes for a broad spectrum of possible disease-causing mutations. © 1996 Oxford University Press Van Orsouw Nathalie J. Molecular Genetics Section, Gerontology Division, Department of Medicine, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA aut Li Daizong Molecular Genetics Section, Gerontology Division, Department of Medicine, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA aut van der Vlies Pieter University of Groningen, Department of Medical Genetics, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands aut Scheffer Hans University of Groningen, Department of Medical Genetics, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands aut Eng Charis Division of Cancer Epidemiology and Control, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA aut Buys Charles H. C. M. University of Groningen, Department of Medical Genetics, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands aut Li Frederick P. Division of Cancer Epidemiology and Control, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA aut Vijg Jan Molecular Genetics Section, Gerontology Division, Department of Medicine, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA aut Human Molecular Genetics Oxford University Press 5/6(1996-06), 755-761 0964-6906 5:6<755 1996 5 hmg https://doi.org/10.1093/hmg/5.6.755 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/hmg/5.6.755 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Van Orsouw Nathalie J. Molecular Genetics Section, Gerontology Division, Department of Medicine, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA aut NATIONALLICENCE 700 1- Li Daizong Molecular Genetics Section, Gerontology Division, Department of Medicine, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA aut NATIONALLICENCE 700 1- van der Vlies Pieter University of Groningen, Department of Medical Genetics, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands aut NATIONALLICENCE 700 1- Scheffer Hans University of Groningen, Department of Medical Genetics, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands aut NATIONALLICENCE 700 1- Eng Charis Division of Cancer Epidemiology and Control, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA aut NATIONALLICENCE 700 1- Buys Charles H. C. M. University of Groningen, Department of Medical Genetics, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands aut NATIONALLICENCE 700 1- Li Frederick P. Division of Cancer Epidemiology and Control, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA aut NATIONALLICENCE 700 1- Vijg Jan Molecular Genetics Section, Gerontology Division, Department of Medicine, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA aut NATIONALLICENCE 773 0- Human Molecular Genetics Oxford University Press 5/6(1996-06), 755-761 0964-6906 5:6<755 1996 5 hmg Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford