Biotechnology
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Ecotilling - Introduction
What is Ecotilling?
Ecotilling is a high throughput, low cost technique for rapid discovery of polymorphisms in natural populations. Ecotilling is similar to Targeting Induced Local Lesions in Genomes, or TILLING® (1), but Ecotilling differs from TILLING in that natural polymorphisms are detected rather than polymorphisms induced through chemical mutagenesis (2). Single nucleotide polymorphisms (SNPs), small insertions and deletions, and variations in microsatellite repeat number can be efficiently detected using the Ecotilling technique on a LI-COR® 4300 DNA Analysis System.
Two-Color Imaging Eliminates False Positives
The LI-COR 4300 DNA Analysis System is uniquely suited for Ecotilling because it uses two-color infrared fluorescence to generate two true gel images during electrophoresis.
Two-color imaging makes identification of polymorphisms very accurate and virtually eliminates false positives. A polymorphism (band) on one gel image is confirmed by the presence of a band in the same lane on the second gel image. The band on the second image is the opposite cut strand and it should be in the expected position (the sum of the molecular weights of the two bands should approximately equal the total size of the amplicon). This unique imaging method also localizes polymorphisms to within ±10 base pairs, making them easy to identify by DNA sequencing.
SNP Discovery Using The Ecotilling Technique
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Sample DNA from up to eight 96-well plates is pooled and mixed with wild type DNA (768 individuals per run). Up to eight individuals can be pooled for each gel lane depending on species and other factors.
During amplification, a 1 kb region of DNA is labeled with two gene-specific primers. Each primer is labeled with a different IRDye®.
After amplification, heteroduplexes between the wild type and sample DNA are cut at mismatch sites by the endonuclease CEL I. Cut strands are denatured and electrophoresed on a Model 4300 Infrared DNA Analyzer. Separate images are simultaneously generated for each fluorescence channel (IRDye®). |
Polymorphisms in one fluorescence channel can be confirmed by the presence of the opposite cut strand in the same lane in the second fluorescence channel (eliminates false positives). Empty lanes (just top and bottom bands) indicate no genetic variation in any of those eight individuals. The eight individuals in lanes with bands can be rerun in separate lanes or sequenced to find the individual(s) with genetic variations. |
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Rapid Haplotyping Using The Ecotilling TechniqueFor rapid haplotyping, 96 individuals can be loaded in separate gel lanes and compared against a reference DNA in each lane. Bands at the same position in different lanes indicate the same haplotype.
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| References 1. Colbert, T., Till, B.J., Tompa, R., Reynolds, S., Steine, M.N., Yeung, A.T., McCallum, C.M., Greene, E.A., Comai, L., and Henikoff, S. 2001. Throughtput Screening for Induced Point Mutations. Plant Physiology 126: 480-484. 2. Comai, L., Young, K., Till, B.J., Reynolds, S.H., Greene, E.A., Codomo, C.A., Enns, L.C., Johnson, J.E., Burtner, C., Odden, A.R., and Henikoff, S. 2004. Efficient Discovery of DNA Polymorphisms in Natural Populations by Ecotilling. The Plant Journal. 37: 778-786. |
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