Frequently asked questions (FAQ)

 



  1. How can I obtain the DUPLO double mutants?
    The DUPLO mutants will eventually be distributed via NASC (the European Arabidopsis Stock Centre). A test set of 5 double mutants (DUPLO DM's) has been donated in October 2012. You can find the "collection description" at the NASC website. There is NO MTA required. The mutants are available as a set together with the true parental lines. We decided for this option because the parents are different from the "lines" that also contain the used allele in that they might have lost (segregated away) additional insertion alleles. All remaining lines will become available from NASC soon.
  2. How were the gene pairs for double mutant construction selected?
    A couple of criteria were important for the selection: the genes in a pair have to show sequence similarity. A third gene with similar properties must not exist in the genome. For practical reasons the duplicated genes must show enough physical distance in the genome to allow recombination after crossing of the individual DUPLO lines in a given pair. And suitable insertions need to exist for both genes.
  3. Which alleles were selected for the genes in a given pair?
    Only lines from the GABI-Kat and SALK collection were considered. If possible, null alleles for the genes were chosen. Therefore, lines with the predicted insertion within coding regions of the gene were preferably selected. Only in cases in which we have started to work on a gene pair, and the addressed allele can not be confirmed, other alleles with the predicted insertion in transcribed regions (mainly 5' of the coding region or introns) are considered.

  4. How is a "gene hit", a "promoter hit", a "TS2TE hit" and a "CDSi hit" defined in GABI-DUPLO?
    Hits are classified the same way as in GABI-Kat. See the GABI-Kat-FAQ9

  5. What is the genetic background of the double mutants?
    The single mutants are from the GABI-Kat and from the SALK collection of T-DNA insertion lines. The genetic background in both cases is Columbia.
  6. What is the workflow for the generation of the double mutants?
    Ideally the procedure is as follows: the predicted insertions in the single mutants are confirmed in a process similar to the one used in GABI-Kat (more information). Then 12-18 independent T2 plants of each line of a given pair are genotyped to identify the homozygous single mutants. The homozygous single mutants are crossed to generate the double hemizygous double mutant, which is re-confirmed by PCR on the DNA of the plants. After selfing of these plants the double homozygous double mutants are identified by genotyping.
  7. Are GABI-Kat and SALK lines processed differently in DUPLO?
    Yes. GABI-Kat lines can be selected with sulfadiazin (more information). The T-DNA in SALK-Lines usually confers a Kanamycin resistance. Because this resistance is not stable in every case or over several generations, 18 plants of a given SALK line grown without selection are genotyped. Usually 12 plants of a GABI-Kat line selected on sulfadiazin-containing plates are genotyped. Furthermore, the T-DNA in GABI-Kat lines is larger than the T-DNA in SALK lines. Therefore there is a fair chance that an intronic insertion will result in a null allele in case of GABI-Kat lines, which is not so sure for SALK lines. This affects the alleles selected for use in DUPLO pairs.
  8. Where can I get more information about the primers that were used in the confirmation process?
    The answer is given on the Confirmation strategy page. Detailed information about the confirmation primers for confirmed alleles are available via SimpleSearch (or DUPLOdb). You may want to have a look at this example (link to the page in DUPLOdb showing the data for genotyping primers for the allele 637C09/At2g32920 that has been selected for the DUPLO pair 49 (At2g32920 + At1g04980)).
  9. Where can I get more information about the primers that were used in the genotyping procedure?
    Detailed information about the genotyping primers for confirmed alleles are available from DUPLOdb. Follow the link to the "show primer details" page. You may want to have a look at this example.
  10. Why do I occasionally find the same gene in two different pairs and with two different partner genes?
    At the beginning of the project a preliminary prediction of DUPLO pairs has been used. The practical work on the respective pairs had been started at that point. Later an enhanced prediction for the pairs was made in which occasionally certain genes were assigned to new partner genes. In cases where the practical work on the lines only present in the older prediction already progressed quite far, both combinations of DUPLO genes were finished.
  11. How many DUPLO lines will become available?
    This questions needs a longer answer ....
    There are 2108 gene pairs that fulfil our selection criteria for a true pair of exactly two sequence-related genes. Of those, we have detected "useful alleles" for both "parental" genes for about 1270 gene pairs. We refer to these pairs as "DUPLO pairs" (see here and also check the note). In the funding period of the DUPLO project, we managed to fully complete the work for 200 double mutants (DM's). These will become available at NASC soon (written as of 20121220 ... at present it is 5 donated DM's, see this example in DUPLOdb and at NASC). Within GABI-Kat, we will confirm over time the remaining "parent alleles" for the DUPLO pairs (as of 20121220, there are about 660 alleles from about 360 pairs (of the total of 1270 pairs) that have not yet been addressed). In addition, there are  some pairs that are not fully complete or in which one of the alleles turned out not to be a CDSi hit. These "incomplete DM's" will also be donated to NASC.