Nucleic Acids Research Advance Access published online on February 22, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm078
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Molecular Biology |
Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector
Stanford Genome Technology Center, Bio-X, 318 Campus Dr., Stanford, California 94305, USA
*To whom correspondence should be addressed. Tel: +1-650-723-6287; Fax: +1-650-724-5791; Email: simon.fredriksson{at}stanford.edu
Received December 29, 2006. Revised January 23, 2007. Accepted January 24, 2007.
Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the us of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.
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