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Analysis of microsatellites by direct blotting electrophoresis and chemiluminescence detection
Author(s) -
Mekus Frauke,
Dörk Thilo,
Deufel Thomas,
Morral Núria,
Tümmler Burkhard
Publication year - 1995
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501601309
Subject(s) - microbiology and biotechnology , chemiluminescence , polymerase chain reaction , chemistry , southern blot , gel electrophoresis , blot , biotinylation , chromatography , biology , dna , gene , biochemistry
We describe a fast and reliable method for the nonradioactive analysis of microsatellites. For three dinucleotide repeats within the cystic fibrosis transmebrane 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‐meth‐oxyspiro{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.