Amplification of ribosomal RNA sequences
Introduction
Comparisons of rRNA sequences, pioneered by Woese and his colleagues, defined the main lineages in the evolution of microorganisms [42]. An advantage of rRNA sequence comparisons is the generation of an increasingly expanding data base against which newly determined sequences may be compared [5, 23]. Nearly 60,000 16S rRNA sequences are currently available in the Ribosomal Database Project II [23]. Initially, sequences were obtained from well described pure cultures for phylogenetic research. Pace et al. [31] recognized that as the rRNA tree filled in, the data base would serve not only for continued comparison of sequences obtained from pure cultures, but also for comparison of sequences obtained directly from natural microbial communities without needing to grow the representative members in the laboratory. The concept of comparing gene sequences from microbial communities revolutionized microbial ecology. Subsequently, a suite...
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- 1.
* Note: mRNA is very sensitive to RNase activity. Sample treatment is conducted on ice as much as possible. Water, reagents, reagent bottles, microfuge tubes and glass slides should be pretreated with DEPC (diethypyrocarbonate). RNase inhibitor is added into RT-PCR mixture.
- 2.
* Note: As mentioned earlier, other labels can be substituted for DIG (see Table 1 also). Signal detection methods discussed here only pertain the use of DIG-labeled nucleotides or probes.
- 3.
* Note: As mentioned earlier, other labels can be substituted for DIG (see Table 1 also). Signal detection methods discussed here only pertain the use of DIG-labeled nucleotides or probes.
- 4.
* Note: Reverse transcription and PCR amplification are carried out using the GeneAmp® EZ r Tth PCR kit (Perkin Elmer), in which both reactions may be accomplished in a single solution mixture. The downstream primer used for RT-PCR with a higher annealing temperature (>60 °C) is desirable for a two-temperature PCR in which annealing and extension temperatures can be combined into one temperature. As mentioned earlier, other labels can be substituted for DIG (see also Table 1). Signal detection methods discussed here only pertain the use of DIG-labeled nucleotides or probes.
- 5.
* Note: This protocol is developed for in situ linear (nonexponential) amplification of RNA sequences by extension of a single primer with labeled nucleotides. As mentioned earlier, other labels can be substituted for DIG (see also Table 1). Signal detection methods discussed here only pertain the use of DIG-labeled nucleotides or probes.
- 6.
* Note: Reverse transcription or RNA sequences is carried out inside intact bacterial cells using a species-specific primer and thermostable reverse transcriptase. No actual PCR amplification is involved in this protocol.
- 7.
* Note: As mentioned earlier, other labels can be substituted for DIG (see also Table 1). Signal detection methods discussed here only pertain the use of DIG-labeled nucleotides or probes.
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Devereux, R., Wilkinson, S.S. (2004). Section 3 update - Identification and classification of microbes using DNA and RNA sequences. In: Kowalchuk, G.A., de Bruijn, F.J., Head, I.M., Akkermans, A.D., van Elsas, J.D. (eds) Molecular Microbial Ecology Manual. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-2177-0_3
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