A simple and rapid electrophoresis method to detect sequence variation in PCR-amplified DNA fragments
A simple and rapid electrophoresis method to detect sequence variation in PCR-amplified DNA fragments
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DOI：
10.1093/nar/23.23.4928
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发表时间：
1995-12-11
影响因子：
14.9
通讯作者：
Muyzer, G
中科院分区：
文献类型：
--
作者：
Wawer, C;Ruggeberg, H;Muyzer, G
Several methods have been described to detect sequence variation in different, but equal-sized DNA fragments. Among them are denaturing gradient gel electrophoresis (DGGE)(1), temperature gradient gel electrophoresis (TGGE)(2), and single-strand conformational polymorphism (SSCP)(3). All these methods utilize polyacrylamide gels, need special equipment, and require pre-experiments to determine the optimal electrophoretic conditions. Another limitation is that only relatively small DNA fragments (up to 500 bp) can be separated with these methods. Here we present a simple, and rapid electrophoresis method which uses agarose gels, does not need special equipment and allows larger DNA fragments to be separated. Electrophoresis is performed in agarose gels containing the DNA ligand bisbenzi-mide, to which long chains of polyethylene glycol (PEG) 6000 are covalently coupled (4). The dye bisbenzimide binds preferen-tially to A+ T sequence motifs in the DNA (5). Therefore, being loaded with the long PEG chains, the A+ T-rich DNA sequences are retarded in the gel relative to the sequences which are low in A+ T content, and so separation is achieved. The principle of this method has been known for 15 years (4), but has only been used incidentally (6).We have extended the use of this method in separating PCR-amplified DNA fragments of the [NiFe] hydrogenase gene from different sulfate-reducing bacteria. Two sets of primers were used to amplify fragments of-440 and 1440 bp, respectively (7). The PCR products were electrophoresed in agarose gels with and without the bisbenzimide/PEG dye in parallel. Figures 1 A and 2A clearly show that PCR products obtained from different bacteria are difficultto separate from each other in agarose gelslacking the bisbenzimide/PEG, but can in gels containing the dye (Figs lB and 2B). Only partial sequence data of the amplified fragments are available (Wawer and Muyzer, unpublished data). However for Desulfovibrio gigas (9) and two other Desulfovibrio strains (10, 11) complete sequences have been published. These data, with sequence similarities ranging between 70 and 90%, indicate that differences in AT content of< 1% have been resolved. A better separation, especially for longerDNA fragments, can be achieved by extending the electrophoresis time. However, because the bisbenzimide/PEG dye slowly migrates in the opposite direction to the DNA, a reservoir of gel containing the DNA ligand in front of the fragments will be necessary. Furthermore, immersion of the gels with electrophoresis buffer, which is common in agarose gel electrophoresis, should be avoided to prevent diffusion of the dye out of the gel. For these reasons,
