Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extract...Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.展开更多
An oligonucleotide primer set (PaF/PaR) was designed for polymerase chain reaction (PCR) amplification of an approximately 1. 2kb fragment DNA from Paulownia Witches’ broom(PaWB) MLO on the basis of 16S rRNA sequence...An oligonucleotide primer set (PaF/PaR) was designed for polymerase chain reaction (PCR) amplification of an approximately 1. 2kb fragment DNA from Paulownia Witches’ broom(PaWB) MLO on the basis of 16S rRNA sequence from O-MLO. Amplification of a 1. 2kb DNA fragment from PaWB-MLO-infected plants DNA in total nucleic acid extrats of the host plant Paulownlh spp., but no l. 2kb DNA fragment was observed when healthy DNA was used as a template. The study showed that the PCR method was able todetect PaWB-MLO in as little as 9. 4pg of total DNA from infected Paulernia tissue culture plantlets.展开更多
基金This work was funded by the Special Scientific Foundation of Guangdong Province (No. A305030301)was partly supported by a grant from KLFEE, Ministry of Agriculture (2003-04).
文摘Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.
文摘An oligonucleotide primer set (PaF/PaR) was designed for polymerase chain reaction (PCR) amplification of an approximately 1. 2kb fragment DNA from Paulownia Witches’ broom(PaWB) MLO on the basis of 16S rRNA sequence from O-MLO. Amplification of a 1. 2kb DNA fragment from PaWB-MLO-infected plants DNA in total nucleic acid extrats of the host plant Paulownlh spp., but no l. 2kb DNA fragment was observed when healthy DNA was used as a template. The study showed that the PCR method was able todetect PaWB-MLO in as little as 9. 4pg of total DNA from infected Paulernia tissue culture plantlets.
