PMID: 8586058Oct 1, 1995Paper

Analysis of microsatellites by direct blotting electrophoresis and chemiluminescence detection

Electrophoresis
Frauke MekusB Tümmler

Abstract

We describe a fast and reliable method for the nonradioactive analysis of microsatellites. For three dinucleotide repeats within the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the separation of polymerase chain reaction (PCR) products generated with biotinylated primers on a direct blotting electrophoresis system and subsequent chemiluminescence detection is shown. In direct blotting electrophoresis, the separation of DNA fragments depended linearly on size. The reproducible resolution allowed reliable assignment of allele lengths to a given signal. The nonradioactive detection protocol was advantageous compared to radioactive methods: samples could be analyzed within one day due to the fast signal development by 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'- chloro)tricyclo[3.3.1.1.3,7]decan]-4-yl)phenylphosphate disodium salt (CSPD). Variation of exposure times enabled differentiation between major bands and byproducts of comparable intensity that are due to the slippage of the Taq polymerase during PCR amplification.

References

Aug 1, 1992·Trends in Genetics : TIG·C M HearneJ A Todd
Aug 1, 1987·Analytical Biochemistry·S Beck
Dec 1, 1994·Journal of Medical Genetics·G R TaylorR F Mueller

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Citations

Oct 19, 2004·Human Genetics·Margit RitzkaBurkhard Tümmler
Apr 23, 2004·BMC Genetics·Nikoletta CharizopoulouBurkhard Tümmler
Mar 31, 2006·BMC Genetics·Nikoletta CharizopoulouBurkhard Tümmler
Apr 11, 2008·BMC Genetics·Balázs TóthBurkhard Tümmler
Dec 7, 2020·Molecular Therapy : the Journal of the American Society of Gene Therapy·Kerstin BrinkertAntje Munder
Jun 15, 1997·Analytical Chemistry·D J AndersonK A Davis

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