Differentiation of bacterial strains by thermal gradient gel electrophoresis using non-GC-clamped PCR primers for the 16S-23S rDNA intergenic spacer region.
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.
Yasuda
Michie
M
Department of Biology, University of Massachusetts Boston, 100 Morrissey Blvd., Boston, MA 02125, USA. michie.yasuda@umb.edu
Shiaris
Michael Peter
MP
D015373:Bacterial Typing Techniques; D016172:DNA Fingerprinting / Q000379:methods; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D020862:GC Rich Sequence / Q000235:genetics*; D006090:Gram-Negative Bacteria / Q000145:classification* / Q000235:genetics; D006358:Hot Temperature; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S