Multimerization of restriction fragments by magnesium-mediated stable base pairing between overhangs: A cause of electrophoretic mobility shift

Hideki Tagashira, Mitsunori Morita, Takashi Ohyama

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

The electrophoretic mobility shift assay (EMSA) or simply the "gel shift assay" is one of the most sensitive methods for studying the ability of a protein to bind to DNA. EMSAs are also widely used to investigate protein- or sequence-dependent DNA bending. Here we report that electrophoresis using physiological concentrations of Mg2+ can cause a mobility shift of restriction fragments in nondenaturing polyacrylamide gels as the overhangs form stable base pairs. This phenomenon was observed at even 37 °C. The retardation was, however, more pronounced at low temperatures, where a three-nucleotide overhang 5′-GAC also caused a mobility shift. The stability of the pairing was generally high when the overhangs of four nucleotides display high GC content, while the mobility shift caused by 5′-AATT was greater than those caused by 5′-GATC, 5′-TCGA, and 5′-CTAG. This observation should be taken into account to avoid misinterpretation of the data when the EMSA, especially the circular permutation gel mobility shift assay, is performed using a running buffer that contains Mg2+ ions. The stable adhesion between short overhangs may present an important basis for genome stability and many genetic processes occurring in living cells.

Original languageEnglish
Pages (from-to)12217-12223
Number of pages7
JournalBiochemistry
Volume41
Issue number40
DOIs
Publication statusPublished - 2002 Oct 8
Externally publishedYes

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Electrophoretic mobility
Electrophoretic Mobility Shift Assay
Base Pairing
Magnesium
Assays
Nucleotides
Gels
Genetic Phenomena
Genomic Instability
Base Composition
Electrophoresis
DNA
Buffers
Proteins
Ions
Temperature
Adhesion
Genes
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

Multimerization of restriction fragments by magnesium-mediated stable base pairing between overhangs : A cause of electrophoretic mobility shift. / Tagashira, Hideki; Morita, Mitsunori; Ohyama, Takashi.

In: Biochemistry, Vol. 41, No. 40, 08.10.2002, p. 12217-12223.

Research output: Contribution to journalArticle

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