Yasuda Michie, Shiaris Michael Peter
Department of Biology, University of Massachusetts Boston, 100 Morrissey Blvd., Boston, MA 02125, USA.
FEMS Microbiol Lett. 2005 Feb 1;243(1):235-42. doi: 10.1016/j.femsle.2004.12.011.
The method for DNA fingerprinting of the 16S-23S rDNA intergenic spacer region was modified to increase resolution of bacterial strains by thermal gradient gel electrophoresis (TGGE) analysis. By utilizing the high melting temperature region of the tRNA gene located in the middle of the 16S-23S rDNA intergenic spacer region as an internal clamp for TGGE, multiple melting domain problems were solved. PCR primers lacking a stretch of GC-rich sequences (GC-clamp) amplified the intergenic spacer region more efficiently than GC-clamped primers. Therefore, PCR artifacts were avoided by using low, 17-cycle, PCR. The method was successfully applied to diverse bacterial species for strain differentiation by TGGE without requiring a special PCR primer set.
对16S - 23S rDNA基因间隔区DNA指纹图谱分析方法进行了改进，通过热梯度凝胶电泳（TGGE）分析提高细菌菌株的分辨能力。利用位于16S - 23S rDNA基因间隔区中间的tRNA基因的高解链温度区域作为TGGE的内部夹钳，解决了多个解链结构域问题。缺少富含GC序列片段（GC夹）的PCR引物比GC夹引物更有效地扩增基因间隔区。因此，通过采用低循环数（17个循环）的PCR避免了PCR假象。该方法成功应用于多种细菌物种，通过TGGE进行菌株区分，无需特殊的PCR引物组。
