Differentiation of bacterial strains by thermal gradient gel electrophoresis using non-GC-Clamped PCR primers for the 16S–23S rDNA intergenic spacer region

Abstract

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.

The CTLA4 -819 C/T and +49 A/G dimorphisms are associated with Type 1 diabetes in Egyptian children

“…[21] The PCR product (20 μl) was mixed with 20 μl of 4 M urea and 0.05% methylene blue, heated at 95°C for 5 min, and 50°C for 20 min. Samples were loaded into 1 mm polyacrylamide gels (5%) covalently bound to polyethylene gel support films (BioRad, Hercules, CA, USA) equilibrated with 1.25× TBE buffer.…”

Microbial Typing by Machine Learned DNA Melt Signatures

“…We did observe identical intraspecies melt profiles based on very limited number of strains for several species in our test panel. However, previous studies had targeted ITS for strain differentiation within one bacterial species 25 26 . Although curve difference is more prominent between species, ITS was shown to have enough intraspecies sequence and copy number variation to produce strain specific melt curves 27 .…”

Differentiation ofBorrelia burgdorferi sensu lato species by temperature gradient gel electrophoresis

“…TGGE was initially developed for a separation of DNA duplexes of the same length, which differ in only a single or few nucleotide sequences [20]. In our study, we have adjusted TGGE for a separation of intergenic space region, with several nucleotide differences between species as well as within species, similar as described by Yasuda and Shiaris [30] by TGGE of 16S-23S rDNA intergenic space region, from various cultivable bacterial species, except that 5S-23S intrgenic space region of B. burgdorferi sensu lato has no coding sequences, neither tRNA nor adenosine and thymidine nucleosides (AT)-or GC-rich regions [21]. With the TGGE method we were able to separate PCR products of 5S-23S intergenic space region and delineate B. afzelii, B. garinii, and B. burgdorferi sensu stricto (Fig.…”

“…In this respect, polymorphism of the 5S-23S intergenic space region was taken into consideration. Polymorphic genetic region has within a given isolate several allelic variants, due to point mutations [21], with multimelting domains [30,32]. Therefore, thermal gradient, which is steep enough, is needed, as established by Gelfi et al [28], so that majority of alles of the same species melt at the same rate and travel together during electrophoresis all the time forming a single, speciesspecific DNA band on the gel.…”