The goal of ATP is to rapidly deliver to the Arabidopsis community EMS-induced mutations in requested genes.  For a description of our high-throughput reverse genetic screens used in TILLING, please refer to Till et al., 2003. Below is a brief description of the steps involved in TILLING an Arabidopsis gene through ATP.  We have outlined what will be done by ATP and what is required of the researcher making the request.  To avoid inefficient uses of our resources and of your time, we ask that you read the following description and cautionary notes very carefully.

 

TILLING: A five step process

1. You decide whether your gene is worth TILLING.

2. You find the best the region to be targeted and place your order.

3. ATP screens the region for mutations.

4. ATP sequences the mutation and enters it in our public database.

5. ATP sends you a mutant report and you order seed.

 

STEP 1:  You (the user) decide whether your gene is a good candidate for TILLING

For each TILLING request, we will screen DNAs from approximately 3000 M2 plants harboring EMS-induced mutations.  As EMS causes G/C to A/T changes, TILLING can provide an allelic transition mutation series.

 

”I have an insertion in my gene but the knockout phenotype is lethal.”

TILLING can provide the sub-lethal phenotypes you want.

 

“The knockout phenotype is interesting.”

TILLING can provide an allelic series that may help you better ascertain the function of your gene.

 

“I have an insertion in my gene that knocks out gene function but my plants have no phenotype.”

TILLING is not for you. In this scenario, a gain-of-function mutation is needed to investigate the potential in vivo role of this gene.  The large majority of phenotypes arising from our populations will cause full or partial loss of function.

 

”I have a candidate gene and I want to know the knockout phenotype.”

There are very good reasons why you should start by insertional mutagenesis rather than by TILLING. First, only a small percentage of EMS-induced mutations will yield a change likely to truncate the protein (~5%). Second, the Arabidopsis community has access to excellent insertional mutagenesis resources. Third, if a knockout mutation causes no phenotype, then the TILLING allelic series is not expected to either.

 

STEP 2: You choose a region of your gene to TILL using the CODDLE website, and place your order. 

ATP targets the requested gene by PCR using gene-specific primers. As our target fragment length is 1.5 kilobase (kb), and the average Arabidopsis gene is about 3-4 kb, it is necessary for the researcher to choose the best region to TILL.  Following this, gene-specific primers must be chosen to amplify this region.  The ATP team has developed a series of computational tools to guide you to the best region to TILL, choose your primers, and allow you to place your order.

 

There are three important components necessary for the optimal TILLING order: 1) a good gene model (intron/exon positions), 2) a good protein sequence homology model, and 3) a good PCR primer pair.  These choices are facilitated by the CODDLE input utility , which accepts genomic, cDNA and/or protein sequences from your own files or via links from public databases.  It then automatically searches for homology information that will help identify regions that are likely to be important for protein function.  Please be patient, as this process might take several minutes .  You will then be provided with options that allow you to examine and improve the homology model.  When you choose CODDLE, be sure to select “TILLING in Plants” as your mutation method (otherwise you will not be able to place an order).  CODDLE then provides analyses of your region, choosing by default the region that it considers the most likely to provide the highest percentage of deleterious lesions in the protein.  This should be advantageous regardless of whether a strong or weak phenotype is desired, although you are encouraged to explore and select other regions if you so desire.  Once the region to TILL has been selected, candidate primer pairs are presented by Primer3 for you to choose from.  CODDLE then evaluates the region encompassed by the chosen primer pair as to its suitability for TILLING and connects you to the TILLING order form.

 

STEP 3: We (ATP) screen our mutagenized population for induced mutations (Till et al., 2003). 

Once we have received your primers from MWG Biotech, your mutational screen will enter our queue. Screens are done on plates containing DNAs pooled 8-fold (DNAs from 8 individuals are mixed).  Once mutant pools are identified, we create a “multiple individual plate” containing DNAs from each of the 8 M2 plants potentially harboring the mutation.  Because we work on a 96-well format, we can detect a maximum of 12 mutations in individual plants in this screen.

 

STEP 4:  We (ATP) sequence each mutation and enter it in our database.

 

STEP 5:  We (ATP) send you a mutation report and you order seed.

If the allelic series from a suitable gene model meets our minimum standard (95% expectation that at least one mutation is damaging to the protein - see User fees), a report describing each mutation is automatically delivered to you. The report identifies the base change, the individual M2 plant from which the DNA was derived, and whether it was homozygous or heterozygous. You will also be provided with a link to the PARSESNP analysis of your mutants. Based on information that you had provided to CODDLE, PARSESNP returns maps of your mutations and algorithmic estimates of how much each mutation is deleterious to the protein. PARSESNP also identifies restriction sites, if any, that distinguish mutant from wild-type.  A direct link from the mutant report to ABRC for each mutant stock facilitates your ordering the seed.

 

With the move to full-cost recovery on October 15, 2005, we ended our 6-month automatic data release policy, but rather now consider data release to be a user option. If this option is chosen, your results will be made available for searching at our Web site and submitted to the TAIR mutation and polymorphism database. This policy does not apply to other plant species, which are currently supported by our NSF PGRP grant, and for which the 6-month data release policy remains in place. Publications that describe studies using STP mutants should cite Till et al., 2003.

 

Is your gene a good TILLING candidate? If so, click to start CODDLing!

Last updated: October 15, 2005

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