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