Targeting Induced Local Lesions In Genomes (TILLING) for Plant Functional Genomics

Targeting Induced Local Lesions In Genomes (TILLING) for Plant Functional Genomics Claire 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

The Authors • 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 • 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

Honorable Mention • 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

Aim of the Paper • 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

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 Overview Heteroduplex 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

Targeting Induced Local Lesions In Genomes (TILLING) for Plant Functional Genomics