Explorative screening of complex microbial communities by real-time 16S rDNA restriction fragment melting curve analyses.
We have developed restriction fragment melting curve analyses (RFMCA), which is a novel method for the real-time analysis of microbial communities. The major advantage of RFMCA compared to, for example, terminal restriction fragment length polymorphism (T-RFLP) or temperature/denaturing gradient gel electrophoresis (TGGE/DGGE) is that the physical separation of DNA fragments is avoided. The RFMCA detection is done by melting point analyses in closed tube systems, which enables high-throughput applications. The robustness of RFMCA was demonstrated by analyzing both mixtures of known samples and the microbial communities in the cecal content of poultry. Our conclusions are that RFMCA is robust, gives a relatively high resolution, and has the potential for high-throughput explorative screenings of microbial communities and large clone libraries.
Rudi
Knut
K
MATFORSK Norwegian Food Research Institute, As, Norway. knut.rudi@matforsk.no
Skånseng
Beate
B
Drømtorp
Signe Marie
SM
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D003199:Computer Systems; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D005243:Feces / Q000382:microbiology*; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D015203:Reproducibility of Results; D015183:Restriction Mapping / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D044366:Transition Temperature
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S