Cancers arise from the accumulation of multiple mutations in genes regulating cellular growth and differentiation. Identification of such mutations in numerous genes represents a significant challenge in genetic analysis, particularly when the majority of DNA in a tumor sample is from wildtype stroma. To overcome these difficulties, we have developed a new type of DNA microchip that combines polymerase chain reaction/ligase detection reaction (PCR/LDR) with 'zip-code' hybridization. Suitably designed allele-specific LDR primers become covalently ligated to adjacent fluorescently labeled primers if and only if a mutation is present. The allele-specific LDR primers contain on their 5'-ends 'zip-code complements' that are used to direct LDR products to specific zip-code addresses attached covalently to a three-dimensional gel-matrix array. Since zip-codes have no homology to either the target sequence or to other sequences in the genome, false signals due to mismatch hybridizations are not detected. The zip-code sequences remain constant and their complements can be appended to any set of LDR primers, making our zip-code arrays universal. Using the K-ras gene as a model system, multiplex PCR/LDR followed by hybridization to prototype 3 x 3 zipcode arrays correctly identified all mutations in tumor and cell line DNA. Mutations present at less than one per cent of the wild-type DNA level could be distinguished. Universal arrays may be used to rapidly detect low abundance mutations in any gene of interest.
Bibliographical noteFunding Information:
We thank Michael Wigler, Donald Bergstrom, Phillip Paty, Eric Spitzer, Leo Furcht, and Matthew Lubin for critical comments and helpful discussions, and express gratitude to our collaborators at PE Biosystems, Michael Albin, Emily Winn-Dean, and Andy Blasband for encouragement and for assistance with spotting arrays. Antonio Picone, Marilyn Khanna, and Monib Zirvi are acknowledged for providing tumor and cell line DNA samples, and for expert technical assistance, and Herman Blok and Maria Kempe are thanked for significant experimental contributions in exploratory stages of this project. Support for this work was provided by the National Institute of Standards and Technology (1995-08-0006F) and the National Cancer Institute (P01-CA65930).
- DNA hybridization
- Ligase detection reaction
- Single nucleotide polymorphism (SNP)
- Thermostable DNA ligase
- Zip-code addressing