Probe Match FAQ
Overview
This FAQ contains information related to using the online Probe Match function. Please direct all corrections and additions to RDP-II General Support (rdpstaff@msu.edu). This FAQ can be found on the Web at http://www.cme.msu.edu/RDP/docs/probe_match_faq.html. Information in this FAQ is primarily based on e-mail received via the RDP-II Support page linked at the bottom of the analysis form.
    Frequently Asked Questions
  1. Q: What does Probe Match do?
    A: Probe Match helps to evaluate a specified probe sequence with rRNA sequences in the RDP database. However, it will not design an oligonucleotide probe for you; it will indicate which RDP database sequences will match your specified probe sequence.

  2. Q: As suggested by the email form [sic] your server, I am notifying you about an error as follows. Do you have any suggestions how to sort it out?
    A: This error message is from the old Illinois RDP site, and its purpose was to alert us to a potential problem if the server was not functioning. It is of little value in actually determining exactly what prevented your probe sequence from being processed. I ran your probe at the MSU RDP-II site and found no matches. It is possible that the probe sequence needs to be flipped, that is, switched from the 5'->3' direction to the 3'->5' direction. Another suggestion would be to relax the search criteria.

    I suggest that you use the MSU RDP-II Probe Match command. The RDP-II Online Analyses URL is http://www.cme.msu.edu/RDP/html/analyses.html and then choose Probe Match.



  3. Q: I would like to design 4 specific primers for V1 regions on 16S rRNA of 4 different bacteria with least possible alignments with other 16s rRNAs from other bacteria. The purpose is detection of bacteria with specific primers. I was wondering if you could give me some hints and information about softwares available for this purpose on the net and if there are instruction books for such kind of work. Is there somewhere where you can differentiate between U-regions and V-regions on 16S rRNA? and is there somewhere as well where you can find specific primer sequences for 16S rRNA for different bacteria?
    A: Several sources are available for looking at primers for the 16S rRNA. I'm aware of no WWW site which will do the designing of the primer for you; several will tell you the areas of the rRNA which are variable or conserved. One WWW site is http://bioc-www.uia.ac.be/u/yvdp/. Another is Robin Gutell's site: http://pundit.icmb.utexas.edu/. Choose 16S secondary structures, and then the Information link. Other WWW sites or resources are in the PCR primers section of the Links file or in Helpful References file.

  4. Q: Do you have any suggestions to find a good empiric Td value?
    A: A reference which discusses Tm and Td values is: Wetmur, James G. "DNA Probes: applications of the principles of nucleic acid hybridization" Critical Reviews in Biochemistry and Molecular Biology 26(3/4): 227-259 (1991).

  5. Q: We would like to detect E.coli K12 cells by ISH with our Facs, and we need a probe which is specific for this strain. We have visited the RDP project Web site, and we have found two sequences (6176 and 6205; from sub-tree 2.14.3.15.2) corresponding to the E.coli K12 strain. How can we proceed to design a probe specific for our strain from these sequences?
    A: The RDP does not provide complete services for the design of specific primers. If you know the sequence of a primer, our Probe Match program is very useful to confirm that your primer sequence will hit E.coli K12. Just type your sequence in and it will generate a list of all the organisms in the database (over 20,000 rRNA sequences) that contain the target site of this primer. If you expect it to hit a large number of sequences - all the bacteria for example - it would be wise to ask the program what your primer doesn't hit because Netscape will crash if one tries to create a results table larger than about 2.4 MB.

    We don't have a list of primer/probe sequences, so searching the literature for primers used by others in E.coli K12 cells might be a productive process. You would then want to the RDP-II Probe Match to test any found primer sequences. Also, you might try the Technical University of Munich's web site http://soul.mikro.biologie.tu-muenchen.de/ORS/

    The Munich group also offers the ARB software package, which includes a Primer/Probe Design program that allows one to search for primers/probes that will hit only a chosen phylogenetic group. That URL is: http://www.mikro.biologie.tu-muenchen.de/pub/ARB/

    You could also look at OPD which is at: http://www.cme.msu.edu/OPD/ to see if a probe for E.coli K12 is specified there.

    Another suggestion is to use the RDP-II site, the Alignment Slices command. Here you will obtain the sequences for specified organisms, however, you'll have to identify the probe sequences yourself. It wasn't clear whether you saw the E.coli K12 sequences from the RDP or RDP-II site. The Alignment Slices command will consolidate the sequences from groups of organisms.



  6. Q: I am about to order some primers for 16s rDNA amplification and I was wondering if you could please tell me what are latest, best universal primers to use and a reference (citation) if possible.
    A: Taken from: Lane, D. J. (1991). 16S/23S rRNA sequencing. Nucleic acid techniques in bacterial systematics. E. Stackebrandt and M. Goodfellow, eds. New York, NY, John Wiley and Sons: 115-175.
    27f5'AGAGTTTGATCMTGGCTCAG>
    342r5'CTGCTGCSYCCCGTAG>
    357f5'CTCCTACGGGAGGCAGCAG>
    519r5'GWATTACCGCGGCKGCTG>
    530f5'GTGCCAGCMGCCGCGG>
    1100r5'GGGTTGCGCTCGTTG>
    1114f5'GCAACGAGCGCAACCC>
    1392r5'ACGGGCGGTGTGTRC>
    1406f5'TGYACACACCTCCCGT>
    1492r5'TACGGYTACCTTGTTACGACTT>
    1525r5'AAGGAGGTGWTCCARCC>
    These represent a small set of published primers. Many variations of these primers and primers targeted to other sites have been published over the years. As more and more sequences have been generated, it has become clear that these "universal" primers should be termed "general" primers, since variation exists even in the most highly conserved regions of the molecule. No single, non-degenerate primer can be expected to target every sequence in a sample, although it may target most of them.


Last updated on 09/13/1999.