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Production of the Model 4300 has been discontinued as of January 1, 2014.
Service and support may become limited. Please contact your local sales office.

Advantages

TILLING Requirements:

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 TILLING1. On gel images produced by an instrument with high sensitivity, new bands resulting from mutations are easily identified.

TILLING Workflow:

Workflow

Eliminating False Positives:

Till1

Figure 1. Data courtesy of the Arabidopsis TILLING project, Seattle, Washington.

Generating High Quality TILLING Images

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.

Two-Color Imaging Eliminates False Positives

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.

High Sensitivity Detection:

Easy Mutation Localization

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 occurred2.

Infrared Detection for Highest Sensitivity

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.

High Throughput Screening:

Efficient Mutation 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*:

  • 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.

In the lab, the 4300 System is fast and efficient:

  • 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

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 researchers3.

  • *Results are dependent on species and other factors.
  • 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 Throughput Screening for Induced Point Mutations. Plant Physiology 126: 480-484.

  • 2. Henikoff, S., and Comai, L. 2003. Single-Nucleotide Mutations for Plant Functional Genomics. Annual Review of Plant Biology. 54:15.1-15.27.

  • 3. 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-Throughput TILLING. Genome Research 13:524-530.



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