1. 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

2. 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

3. 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

4. The Authors Elizabeth A. Greene Luca Comai
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

5. 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

6. 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

7. 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

8. 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

9. 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

10. TILLING Overview Pooling of Samples
An individual plate has 64 wells in use, each with DNA from a single unique individual

11. 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

12. 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...

13. 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

14. 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

15. TILLING Overview
Heteroduplex Formation

16. 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

17. 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

18. 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

19. 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.

20. 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

21. LI-COR Gel Image
TILLING Overview

22. 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

23. 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

24. Future of TILLING Detection of polymorphisms C. elegans
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

25. 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

26. 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