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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