A web forum for questions and answers (hopefully!) regarding the Suicide Polymerase Endonuclease Restriction (SuPER) methodology.

Monday, November 28, 2005

SuPER PCR Top 20

UPDATE: Sorry to say, SuPER PCR is no longer in the top 20 from Oct-Dec 2005. However, with your help, perhaps it can reclaim its former glory!

Jason Smith from the University of Florida just sent me an email telling me that the SuPER PCR article was one of the top 20 most requested articles during the period of July - September 2005. Pretty neat!

Thursday, November 10, 2005

Reduce the impact of plastid DNA in root bacterial community analyses

One other thought. The paper below suggests that the impact of plastid DNA in bacterial analyses can be mitigated by employing RNA extraction-reverse transcription-PCR analyses. Since plastids are not particularly active in root cells, the relative contribution of plastids to total RNA in a root extract will be reduced. This probably won't work in leaf extracts!

Nikolausz, M., K. Marialigeti, and G. Kovacs. 2004. Comparison of RNA-and DNA-based species diversity investigations in rhizoplane bacteriology with respect to chloroplast sequence exclusion. J. Microbiol. Methods 56:365–373.

A Technique for Targeted RNA Cutting

I would like to inform anyone who is interested in the SuPER methodology of a similar technique for RNA. This other methodology, Sequence-specific cleavage of SSU rRNA with oligonucleotides and RNase H, is similar conceptually. Extracted RNA is incubated with a specific DNA probe and RNase H is added to the mixture. RNase H will cut an RNA:DNA hybrid, but will not damage single-stranded RNA. Thus, a specific primer can be used to cut an RNA target, leaving non-target molecules intact. These intact molecules can be recovered and used as a template for downstream applications such as reverse transcription. Dr. Uyeno et al. took the further step of using the technique to quantify microbial populations. A very clever technique indeed.

Yutaka Uyeno, Yuji Sekiguchi, Akiko Sunaga, Hiroki Yoshida, and Yoichi Kamagata (2004). Sequence-Specific Cleavage of Small-Subunit (SSU) rRNA with Oligonucleotides and RNase H: a Rapid and Simple Approach to SSU rRNA-Based Quantitative Detection of Microorganisms. Appl. Environ. Microbiol. 70:3650-3663.