Analysis of microsatellites by direct blotting electrophoresis and chemiluminescence detection

F. Mekus, T. Dork, T. Deufel, Nuria Morral, B. Tummler

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1886-1888
Number of pages3
JournalElectrophoresis
Volume16
Issue number10
StatePublished - 1995
Externally publishedYes

Fingerprint

Chemiluminescence
Polymerase chain reaction
Luminescence
Electrophoresis
Microsatellite Repeats
Taq Polymerase
Dinucleotide Repeats
Cystic Fibrosis Transmembrane Conductance Regulator
Polymerase Chain Reaction
Regulator Genes
Reaction products
Amplification
Byproducts
Genes
Alleles
DNA
phenylphosphate
1,2-dioxetane
3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane

Keywords

  • Chemiluminescent detection
  • Direct blotting electrophoresis
  • Microsatellite analysis

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Analysis of microsatellites by direct blotting electrophoresis and chemiluminescence detection. / Mekus, F.; Dork, T.; Deufel, T.; Morral, Nuria; Tummler, B.

In: Electrophoresis, Vol. 16, No. 10, 1995, p. 1886-1888.

Research output: Contribution to journalArticle

Mekus, F, Dork, T, Deufel, T, Morral, N & Tummler, B 1995, 'Analysis of microsatellites by direct blotting electrophoresis and chemiluminescence detection', Electrophoresis, vol. 16, no. 10, pp. 1886-1888.
Mekus, F. ; Dork, T. ; Deufel, T. ; Morral, Nuria ; Tummler, B. / Analysis of microsatellites by direct blotting electrophoresis and chemiluminescence detection. In: Electrophoresis. 1995 ; Vol. 16, No. 10. pp. 1886-1888.
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