4300 IMAGER
Applications for the 4300 Analysis System
TILLING
Application Overview
Rapid acquisition of genomic sequence data has elevated a new discipline, functional genomics, which focuses on determination of gene function. Reverse genetics methodologies are an important part of functional genomics.
Traditional reverse genetic methods, such as the use of transposons to "knock out" a specific gene, can accurately determine phenotype but require time consuming transgenic or sophisticated tissue culture methodologies. Such "knockout" methods are limiting because the entire gene is knocked out - the effects of partial loss of function of an active gene cannot be observed.
To overcome the limitations of knocking out an entire gene and to expand knowledge of active gene mutations, researchers from Fred Hutchinson Cancer Research Center developed a process for Targeting Induced Local Lesions In Genomes, or TILLING.
Elegantly simple, yet highly efficient, TILLING uses chemical mutagenesis to yield a traditional allelic series of point mutations for virtually all genes. The TILLING process is of particular value for essential genes where sublethal alleles are required for phenotypic analysis. The value of TILLING for genetic research is enhanced by its proven viability for a rapidly growing range of organisms.
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Figure 1
Click here to download TILLING brochure.
Advantages
Tilling Overview
Point mutations of a gene are relatively subtle, making discovery a challenge for the researcher and the detection instrument. Sensitivity of the instrument is critical for detection. The ability to distinguish false positives is of equal importance. Gel images from slab gel electrophoresis have emerged as a data format well suited for TILLING (1). On gel images produced by an instrument with high sensitivity, new bands resulting from mutations are easily identified.
TILLING Requirements:
1 Colbert, T., Till, B.J., Tompa, R., Reynolds, S., Steine, M.N., Yeung, A.T., McCallum, C.M., Greene, Comai, L., and Henikoff, S. 2001. High Throughtput Screening for Induced Point Mutations. Plant Physiology 126: 480-484.
Generating High Quality TILLING Images
Two-Color Imaging Eliminates False Positives
Eliminating False Positives:
The LI-COR 4300 DNA Analysis System is uniquely suited for TILLING because it uses two-color infrared fluorescence detection to generate two true gel images during electrophoresis. Unprocessed image data are critical for TILLING because systems that highly process fluorescence data during detection will likely filter out most, if not all, mutations.
Mutation identification on the 4300 System is very accurate. False positives are virtually eliminated by two-color imaging. Even on the highly reduced gel images shown to the right, bands of mutations are clearly visible (circled red and green). When heteroduplexes created during PCR are cleaved at base mismatches (see TILLING Workflow), the result is two cleavage fragments labeled with different IRDyes. With two color imaging, a true mutation has two mutant bands below the wild type band in the same lane. One band is on the IRDye® 700 infrared dye image and the other on the IRDye® 800 infrared dye image. Additionally, the sum of the molecular weights of the mutant bands in a lane must equal the molecular weight of the wild type band in order for the mutation to be confirmed. Using this two-color detection method, the 4300 System virtually eliminates false positive mutation identifications.
[ABOVE] Figure 3. Data courtesy of the Arabidopsis TILLING project, Seattle, Washington.
Easy Mutation Localization
Infrared Detection for Highest Sensitivity
High Sensitivity Detection:
After mutation identification, imaging programs like Adobe® Photoshop® can be used to determine the lane number where the mutation occurred and the approximate molecular weights of the bands, which confirms the mutation. This technique localizes the mutation within ±10 base pairs, making it easy to identify by DNA sequencing. Localization of the mutation, particularly for heterozygotes, is a clear advantage over methods such as DHPLC and TGCE, which do not indicate where the mutation occurred (3).
The high sensitivity of infrared detection in LI-COR's 4300 System is critical because exonuclease activity in TILLING results in some lost signal. The inherent low background of infrared detection compared to visible detection, combined with LI-COR's IRDye® infrared dyes, results in another key advantage -- wide dynamic range. Wide dynamic range is critical for resolving weak mutation bands along with strong wild type bands. Low infrared background from amplification artifacts makes it easy to resolve mutations with the 4300 System.
3 Henikoff, S., and Comai, L. 2003. Single-Nucleotide Mutations for Plant Functional Genomics. Annual Review of Plant Biology. 54:15.1-15.27.
Efficient Mutation Screening
Up to 750,000 base pairs screened per run.
Up to 2000 samples screened per day.
Up to 2 million base pairs screened per day.
1000 base pairs per sample.
96-well membrane combs load in just a few minutes.
2-4 hour run time (dependent on the size of the amplicon).
Pre-mixed KBPlus gel formulations.
Gels can be reloaded several times.
Proven Performance, Impressive Results
High Throughout Screening:
TILLING can be used as a high throughput screening technique. With the 4300 System as the enabling technology, the following TILLING performance results can be achieved with a single instrument*:
*Results are dependent on species and other factors.
In the lab, the 4300 System is fast and efficient:
The strength of the 4300 System for TILLING is being proven every day in multiple labs around the world. For example, in the first year of operation, the Arabidopsis TILLING Project (ATP) used LI-COR systems to deliver more than 1000 mutations in over 100 genes to Arabidopsis researchers (4).
4 Till, B.J., Reynolds, S.H., Greene, E.A., Codomo, C.A., Enns, L.C., Johnson, J.E., Burtner, C., Odden, A.R., Young K., Taylor, N.E., Henikoff, J.G., Comai, L., and Henikoff, S. 2003. Large-Scale Discovery of Induced Point Mutations With High-Throughtput TILLING. Genome Research 13:524-530.
Technical Resources
Protocols:
Mung Bean Protocol v1.0
LI-COR (9/2008)
4300,protocolsAdobe Photoshop for TILLING v1.0
Jeff Harford (9/2008)
This is a datasheet for using Adobe Photoshop to analyze TILLING/Ecotilling dataUsing Adobe Photoshop to Postprocess LI-COR Images for TILLING applications v1.0
LI-COR (9/2008)
4300,protocolsApplication of TILLING/Ecotilling to Screen for Small Mutations in Mammals v1.0
Kovar, et. al. (9/2008)
Poster presentation on use of 4300 with Tilling and Ecotilling to screen for small mutations in mammals
PubMed
Tilling References
Colbert, T., Till, B.J., Tompa, R., Reynolds, S., Steine, M.N., Yeung, A.T., McCallum, C.M., Greene, Comai, L., and Henikoff, S. 2001.
High Throughtput Screening for Induced Point Mutations. Plant Physiology 126: 480-484.
To see other publications on Tilling click here.
