Summary
The effect of a stacking gel, the pH and cross-linking agent concentration on the resolution and sharpness of PCR amplified VNTR alleles in a vertical discontinuous polyacrylamide gel electrophoresis system was investigated. The experiments show that the use of a low crosslinking agent concentration, a stacking gel and a wide pH difference between the gel buffer and the electrophoresis buffer at the beginning of the electrophoresis resulted in reduced band width and increasing resolution in silver-stained polyacrylamide gels. The importance of sharp DNA fragments is especially emphasized when analyzing multi-allelic DNA loci, that exhibit alleles differing from only few by to few dozen by in length, such as variable number of tandem repeat (VNTR) or short tandem repeat (STR) loci.
Zusammenfassung
Die Auswirkungen eines geschichteten Gels, des pH-Wertes und der Konzentration der Vernetzungs-Substanz auf die Auflösung und Schärfe der Bandendarstellung von PCR-amplifizierten VNTR-Allelen wurden in einem vertikalen diskontinuierlichen Polyacrylamidgel-Elektrophoresesystem untersucht. Die Experimente zeigen, daß die Verwendung einer niedrigen Konzentration der Vernetzungs-Substanz, eines geschichteten Gels und eines großen pH-Unterschiedes zwischen dem Gelpuffer und dem Electrophoresepuffer zu Beginn der Elektrophorese zu einer reduzierten Bandbreite führte, verbunden mit einer zunehmenden Auflösung in silbergefärbten Polyacrylamidgelen. Die Darstellung von DNA-Fragmenten in Form scharfer Banden ist besonders wichtig, wenn multi-allelische DNA-loci untersucht werden, welche Allele aufweisen; die sich im Bereich von wenigen Basenpaaren bis wenigen Dutzend Basenpaaren in der Länge unterscheiden, wie z. B. Loci mit einer variablen Anzahl von Wiederholungseinheiten (VNTR), besonders Loci mit kurzen Wiederholungseinheiten (STR).
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Sajantila, A., Lukka, M. Improved separation of PCR amplified VNTR alleles by a vertical polyacrylamide gel electrophoresis. Int J Leg Med 105, 355–359 (1993). https://doi.org/10.1007/BF01222121
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DOI: https://doi.org/10.1007/BF01222121