Analysis of microsatellites by direct blotting electrophoresis and chemiluminescence detection.
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.
Mekus
F
F
Klinische Forschergruppe Molekulare Pathologie der Mukoviszidose, Medizinische Hochschule Hannover, Germany.
Dörk
T
T
Deufel
T
T
Morral
N
N
Tümmler
B
B
D000483:Alleles; D001710:Biotin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008163:Luminescent Measurements; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D001710:Biotin; D004247:DNA