Why do we isolate DNA not RNA in 16 S rRNA technique for bacteria identification?


closed as off-topic by kmm, AliceD, MattDMo, WYSIWYG Aug 26 '16 at 10:41

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  • $\begingroup$ Please ask only one question at a time. Your other questions have already been answered here and across the web. Please do your own research before asking. $\endgroup$ – MattDMo Aug 24 '16 at 16:32

There are several reasons some of which are fairly obvious:

  1. The most obvious reason is that PCR only works with DNA, because it uses DNA-dependent DNA-polymerase. One can argue that you can use an RNA-dependent RNA-polymerase, but it has several drawbacks, mainly the cost and synthesis accuracy (Lauring et al., 2013, Nature). Since RNA-polymerase itself has higher error rates than DNA-polymerase (Berg et al., Biochemestry 5th edition, 2002) RNA transcripts might already have some variation from the original gene and using a low-fidelity polymerase in PCR will only lead to exponential error accumulation, which reduces identification resolution.
  2. RNA is less stable in aqueous solutions due to the additional -OH group at the 2' position in the ribose, hence RNA extraction and storage is more complicated and expensive in most cases.
  3. In community studies DNA and RNA have different biological implications, hence metagenomics and metatranscriptomics are different fields.
  • $\begingroup$ There are several issues with this answer. (Point 1) RNA sequencing is cheap, easy, and very accurate nowadays. cDNA conversion of RNA, also known as reverse-transcriptase RCR, has been common for quite some time now, with highly accurate RTases being used from many different suppliers. A study from 1988 about HIV is not very current evidence of your thesis, especially in the incredibly rapidly-changing world of mol bio. (Point 2) RNA-Seq instruments are provided by a number of companies, and they all work (initially) with RNA. It is also possible through a variety of methods (...) $\endgroup$ – MattDMo Aug 24 '16 at 16:43
  • $\begingroup$ (...) to get quite good results using nanopore sequencing nowadays. (Point 3) the second part of the sentence is just wrong. Take a sample, throw it in RNeasy, and store it in the freezer indefinitely. Buy a cheap (depending on your budget) Qiagen spin column, purify the RNA at your convenience, aliquot, and store indefinitely at -20 or -80. Cheap and easy. I'm not sure what the point of Point 4 is. $\endgroup$ – MattDMo Aug 24 '16 at 16:46
  • $\begingroup$ @MattDMo thanks for your feedback. 1. I removed the revertase-related statement, though I've found no papers comparing error rates of high fidelity DNA-polymerases and high fidelity revertases (it would be great to get one from you); 2. I've been unaware there are machines, except for nanopore, that directly sequence RNA; as for nanopore, although huge improvements have been made, I haven't seen any reports with accuracy > 0.9 (which is laughable when we speak about identification). 3. I've used that kit myself, that's why I wrote that RNA extraction is more complicated and expensive... $\endgroup$ – Eli Korvigo Aug 24 '16 at 18:38
  • $\begingroup$ ... As with RNA-sequencing, I haven't written that it is impossible or something, I've only said that it is more expensive and complicated than DNA-sequencing. And when you only need to identify something, you go for the cheaper and simpler solution, given equal precision; 4. in community studies RNA-seq is commonly used to distinguish active and dormant microorganisms (though it is a complicated theoretical issue), while DNA-sequencing is used to assess structure and composition at the highest possible resolution; DNA-seq usually uncovers more diversity. $\endgroup$ – Eli Korvigo Aug 24 '16 at 18:47

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