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

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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
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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

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The Authors

• Claire M. McCallum
• Developed technique while a graduate student in
  Dr. Henikoffs 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

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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

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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

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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

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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

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TILLING Overview

• Mutagenesis
• Most important step because if you dont 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

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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

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TILLING Overview

• Pooling of Samples

An individual plate has 64 wells in use, each
with DNA from a single unique individual
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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
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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...

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TILLING Overview

• PCR
• Primers must be carefully selected to ensure that
  they are going to amplify a suitable region
• dont 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

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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

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TILLING Overview
Heteroduplex Formation
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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

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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

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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

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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.

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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

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TILLING Overview

• LI-COR Gel Image

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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

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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

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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

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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

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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