Manuscript Using SuPER-PCR

The SuPER method was recently used to expose the presence of Pseudomonas species in the digestive system of fruit flies. The digestive system is predominantly composed of Enterobacteriaceae and these are detected routinely with bacterial 16S rRNA gene primers. Behar et al. (2008) describe the application of an Enterobacteriaceae-specific SuPER primer to digest away the 16S rRNA genes of these organisms. Standard bacterial PCR was then able to detect Pseudomonas. This was confirmed with specific cultivation of these organisms. A nice study and application of the SuPER method.

Gut bacterial communities in the Mediterranean fruit fly (Ceratitis capitata) and their impact on host longevity

A. Behar, B. Yuval and E. Jurkevitch.

Abstract

Fruit flies (Diptera: Tephritidae) harbor stable bacterial communities in their digestive system, composed mainly of members of the Enterobacteriaceae. However, the Enterobacteriaceae are not the sole community in this habitat. We examined the hypothesis that Pseudomonas spp. form a cryptic community in the gut of Ceratitis capitata, the Mediterranean fruit fly (‘medfly’). Suicide polymerase restriction PCR (SuPER PCR), a novel culture-independent technique, revealed that Pseudomonas spp. form minor, common and stable communities within the medfly's gut. These include P. aeruginosa, a known pathogen of arthropods. Experimental inoculations with high levels of P. aeruginosa reduced the medfly's longevity while inoculations with members of the Enterobacteriaceae extended the fly's life.

Accordingly, we suggest that in addition to their possible contribution to the fly's nitrogen and carbon metabolism, development and copulatory success (as shown in previous studies), the Enterobacteriaceae community within the medfly's gut may also have an indirect contribution to host fitness by preventing the establishment or proliferation of pathogenic bacteria.

Questions regarding template addition and cleanup

QUESTION: I would like to know how much DNA templates should be added to the SuPER reaction mixtures and how much dna templates do you use after the SuPer reaction to perform the normal PCR.

First, there is no specific amount of DNA you should add to the SuPER reaction, and it may take some optimization to get the right amount. We managed well with something like 10-40 ng of genomic DNA in the reactions (25 ul volume) to remove plastids. However, the reaction could probably handle more genomic DNA. At some you will probably overload the reaction and get incomplete digestion. Thus, I recommend you try a range of different amounts of DNA until you find an optimum for your samples. Also, remember that the amount of genomic DNA you add isn’t really the relevant issue, but rather amount of target DNA you’ve added to the system. This gets us to your second question, which I’m afraid to say will also require optimization. The more dominant the template you are trying to remove, the less uncut DNA will remain after the SuPER reaction. If your system is 99% DNA to get cut with the SuPER reaction and you add 100 ng to the reaction, you will only have 1 ng of uncut DNA in 25 ul after the reaction. In such cases I often had to reduce the annealing temperature of the subsequent PCR reaction while keeping magnesium concentrations at 4.0 mM to get reasonable PCR amplification. However, if your target DNA to get cut is 50% of the DNA, you will have 50 ng remaining which will be much easier to amplify.

QUESTION: The second question is why don't you clean you DNA template after the SuPER reaction to avoid inhibition problems in the normal PCR ?

We wanted to minimize the number of steps involved in the reaction, and cleaning up of the DNA inevitably results in a certain loss of DNA. For those reactions in which DNA concentrations are really low after the SuPER reaction I was worried about the inability to amplify the DNA afterwards. Plus, I found that if I reduced the primer concentration and digested the enzyme, I could directly PCR the resulting DNA. You are free to clean up the DNA… there is no theoretical problem with it. If you can get enough DNA (perhaps combining multiple tubes) it probably will be even better.

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!

http://aem.asm.org/misc/Top_20.shtml

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.

Another Restriction Enzyme for SuPER PCR

Dr. Stella Loke, from the University of Sydney, emailed me to tell me that the restriction enzmye TaqIa (TCGA) is superior to Tsp509I. The optimum temperature for the enzmye is 65 degrees, but apparently it can be fully heat inactivated, unlike Tsp509I. This may help avoid the step of adding Proteinase K to the reaction to remove the Tsp509I enzmye.

SuPER PCR

This website is maintained by the authors of the paper "Suicide polymerase endonuclease restriction, a novel technique for enhancing PCR amplification of minor DNA templates". We are hoping to maintain an open forum for people interested in the technique and to offer any assistance, if possible. We would also like to hear people's comments and maintain a list of restriction enzymes and primers that researchers have used successfully.

Please feel to contact either Stefan Green (sjgreen@fsu.edu) or Dror Minz (mail: minz@volcani.agri.gov.il) or leave a message on this site.

Green, S.J. and Minz, D. 2005. Suicide polymerase endonuclease restriction, a novel technique for enhancing PCR amplification of minor DNA templates. Appl. Environ. Microbiol. 71(8):4721-4727.