Skip to main content
Article thumbnail
Location of Repository

High-Throughput Detection of Induced Mutations and Natural Variation Using KeyPoint™ Technology

By Diana Rigola, Jan van Oeveren, Antoine Janssen, Anita Bonné, Harrie Schneiders, Hein J. A. van der Poel, Nathalie J. van Orsouw, René C. J. Hogers, Michiel T. J. de Both and Michiel J. T. van Eijk


Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variants among mutant or natural populations. Current (pre-) screening methods such as TILLING and EcoTILLING are based on the detection of single base mismatches in heteroduplexes using endonucleases such as CEL 1. However, there are drawbacks in the use of endonucleases due to their relatively poor cleavage efficiency and exonuclease activity. Moreover, pre-screening methods do not reveal information about the nature of sequence changes and their possible impact on gene function. We present KeyPoint™ technology, a high-throughput mutation/polymorphism discovery technique based on massive parallel sequencing of target genes amplified from mutant or natural populations. KeyPoint combines multi-dimensional pooling of large numbers of individual DNA samples and the use of sample identification tags (“sample barcoding”) with next-generation sequencing technology. We show the power of KeyPoint by identifying two mutants in the tomato eIF4E gene based on screening more than 3000 M2 families in a single GS FLX sequencing run, and discovery of six haplotypes of tomato eIF4E gene by re-sequencing three amplicons in a subset of 92 tomato lines from the EU-SOL core collection. We propose KeyPoint technology as a broadly applicable amplicon sequencing approach to screen mutant populations or germplasm collections for identification of (novel) allelic variation in a high-throughput fashion

Topics: Research Article
Publisher: Public Library of Science
OAI identifier:
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://www.pubmedcentral.nih.g... (external link)
  • Suggested articles


    1. (1993). A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications.
    2. (2005). A reverse genetic, non-transgenic approach to wheat crop improvement by TILLING.
    3. (2007). Accuracy and quality of massively parallel DNA pyrosequencing.
    4. (2007). Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance.
    5. (2007). Complexity Reduction of Polymorphic Sequences (CRoPS TM): A Novel Approach for Large-Scale Polymorphism Discovery in Complex Genomes.
    6. (2004). Discovery of induced point mutations in maize genes by TILLING.
    7. (2007). DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations.
    8. (2004). Efficient discovery of DNA polymorphism in natural populations by Ecotilling.
    9. (1999). Enzymatic and chemical cleavage methods.
    10. (2005). Genome sequencing in microfabricated high-density picolitre reactors.
    11. (2004). Global impact of mutation-derived varieties.
    12. (2001). Highthroughput screening for induced point mutations.
    13. (2004). In silico screening of a saturated mutation library of tomato.
    14. (2006). Methods for reverse genetic screening in zebrafish by resequencing and TILLING.
    15. (2008). MNUinduced mutant pools and high performance TILLING enable finding of any gene mutation in rice.
    16. (2007). s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis.
    17. (2007). Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis.
    18. (2003). Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis.
    19. (2000). Targeted screening for induced mutations.
    20. (2006). The potential of electrophoretic mobility shift assays for clinical mutation detection.
    21. (1999). The protein truncation test: A review.
    22. (2005). The recessive potyvirus resistance gene pot-1 is the tomato orthologue of the pepper pvr2-eIF4E gene.
    23. (2004). The restriction site mutation (RSM) method: clinical applications.
    24. (2006). TILLING is an effective reverse genetics technique for Caenorhabditis elegans.
    25. (2008). TILLING to detect induced mutations in soybean.
    26. (2005). Tilling without a plough; a new method with application for reverse genetics.

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.