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Targeting Induced Local Lesions In Genomes (TILLING) for Plant
Functional GenomicsClaire M. McCallum, Luca Comai, Elizabeth A. Greene, and
Steven Henikoff (2000) Plant Physiology
Presented by Adam Warner
The Authors
• Steven Henikoff• Basic Sciences division of the Fred Hutchinson
Cancer Research Centre in Seattle Washington
• Currently working to expand TILLING to other organisms
• Believes that TILLING could improve certain crops through gene knockouts and alterations, while not needing to insert foreign DNA. This could alleviate pressure from groups lobbying against GMOs
• Claire M. McCallum• Developed technique while a graduate student in
Dr. Henikoff’s lab
• Was discouraged when trying to create gene knockouts of genes coding for chromomethylases (possible role in silencing)
• Developed TILLING to study role of chromomethylases by creating allelic series of target genes
The Authors
• Elizabeth A. Greene• Currently working in bioinformatics field at the Fred
Hutchinson Cancer Research Centre in Seattle Washington
• Created program to calculate that a gene segment will contain a damaging mutation
The Authors
• Luca Comai• Provided space for growing plants used in
experiments carried out for this paper• Currently a PI on the TILLING project at the
University of Washington
• Bradley Till• Runs the TILLING project at the University of Washington on a day to
day basis
• Provides free workshops to the research community in order to facilitate the use of TILLING for other organisms
• Did not invent TILLING technique
• Provides excellent bus directions to Key Arena for basketball games, complete with map, schedule of times, and much more
• Loves Canadian beer
Honorable Mention
• To introduce a new technique useful for creating an allelic series of gene disruption/knockout to the scientific community
• Raising interest in the the technique to generate new ideas for improvement of TILLING, and expand TILLING to other organisms
• Provide insight into possible uses for TILLING, such as genetic modification of crops
Aim of the Paper
TILLING Overview
• Mutagenesis• First you need to have a mutagenized population
from which to begin the process
• Typically, you want to have a rate of one mutation per 300,000 bp when creating your population.
• A good mutagenesis efficiency lowers costs, but too much mutation causes problems in progeny (lethals, poor growth, higher need for outcrossing later)
• EMS is the mutagen used most often
TILLING Overview
• Mutagenesis• Most important step because if you don’t have a good
population to begin with, the rest of the procedure is a waste
• 50% of mutations are silent• 5% of mutations are truncations• 45% of mutations are missense
– of these missense mutations, approximately 33% change the phenotype
• overall, 10% of mutations cause a phenotypic change
TILLING Overview
• Pooling of Samples• in order to check many samples for a possible mutation,
samples must be pooled
• using the pooling method, 768 different individuals can be screened for a mutation
TILLING Overview
• Pooling of Samples
An individual plate has 64 wells in use, each with DNA from a single unique individual
TILLING Overview• Pooling of Samples
Individual Plate
The Pool plate takes the individual DNA samples from a whole column of an individual plate and puts it into one well. A total of 12 individual plates are pooled this way
TILLING Overview
• Pooling of Samples• In total, the DNA from 8 individuals is in each
well of the 96 well pool plate
• Everything is carefully marked so that if a mutation is detected, the individual plate and column are known
• After pooling, PCR begins...
TILLING Overview
• PCR• Primers must be carefully selected to ensure that
they are going to amplify a suitable region• don’t want to amplify non-coding region
• use of a longer primer and high Tm helps to increase specificity, and decrease noise on the LI-COR gel
• Taq proofreading is not all that important because if something looks like a mutation in step one of procedure, chances of it showing up in step 2 as well are very low
TILLING Overview
• PCR• Approximately 100ng of product is desired so that a
concentration of 10ng/ul is reached• About 45 cycles are required to reach this level • End step of PCR is to denature all DNA present, then
reanneal• this causes a small bubble to form between mismatched
pairs of DNA (where the mutation has occurred) forming a heteroduplex
• Labelling with 2 different dyes occurs in order to facilitate imaging detection process
TILLING OverviewHeteroduplex Formation
TILLING Overview
• Detection of Mutations– DHPLC
• This is the method used originally, but now the enzyme Cel-1 is used
• not as useful for high throughput because of the time required to run a sample
• can detect heteroduplexes with good efficiency, but cannot give good specificity as to where the mutation is in the gene
TILLING Overview
• Detection of Mutations– Cel-1
• derived from celery
• cuts DNA at a mismatch (heteroduplex)
• exact role in cell is not known but may function to cut up single stranded nucleic acids from infecting viruses
• can be overactive at 45ºC and cut at large stretches of AT due to the looser bonds between these pairings
• cuts at 3’ end of mismatch
TILLING Overview
• Cel-1 Digestion• Cel-1 is added to the final PCR products and
cuts at bubbles formed in heteroduplexes
• After digestion, reaction is stopped
• Sephadex beads are used to clean up each sample so that only water and DNA are left
TILLING Overview
• Gel Running• Samples are loaded onto a comb using either a
robot or manually with a pipettor
• Comb is used to load samples onto a LI-COR Gel
• Samples are run until they run completely off the gel
• LI-COR gel running machine detects fluorescent tags on fragments and creates a real time image of the gel as it runs.
TILLING Overview
• Gel Running• Since each fragment should be labelled with the 2
different dyes used, if there is a mismatch and the DNA is cut, two smaller fragments will be present, one labelled green, one red
• These 2 fragments will add up to the same molecular weight as the wild type fragment
• When the gel is analysed, the image showing red labelled fragments and the image showing green labelled fragments will complement
– through this methodology, an almost exact identification of the base pair where the mutation occurred is possible
TILLING Overview• LI-COR Gel Image
TILLING Overview
• Analysis• After finding a mutation, the mutation can be
narrowed down the almost the exact basepair, but it could be one of 8 different individuals because of the pooling process
• The individual plate where the pooled samples came from is rerun with the eventual idea being that each individual gets its own lane on the gel
– this allows for exact identification of the individual that carries the mutation
Results of TILLING
• Allelic Series Created• Due to different mutations causing either
truncations, single amino acid changes, etc, mutations affecting the protein of interest are varied
– this allows for an allelic series which may cause differing phenotypes and allow for greater understanding of protein function than a single knockout could provide
Future of TILLING• Detection of polymorphisms
• detection of mismatches can provide excellent detection of polymorphisms due to the mismatch of different alleles
• C. elegans • Can be used in C. elegans as well as many other
species to create and allelic series for a gene of interest
• Crop Improvement• Allelic series can cause change in protein function that could be
beneficial
• Not having addition of foreign DNA alleviates many worries for consumer groups
Summary
• TILLING is an effective technique to use to gain insight into gene function
• While other techniques have been and are becoming available, TILLING continues to expand to new areas
• TILLING is adaptable to a high throughput environment
• TILLING continues to evolve and improve as a technique
Acknowledgments
• Information and pictures provided by the Fred Hutchinson Cancer Research Centre and LI-COR
• An extensive overview of TILLING was provided by Brad Till
• Thanks to Nick for giving me a short paper that I already knew a decent amount about