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METHODS

Polymorphism Ratio Sequencing: A New Approach for Single Nucleotide Polymorphism Discovery and Genotyping

¹University of California, Berkeley/University of California, San Francisco Joint Bioengineering Graduate Group, Berkeley, California 94720, USA; ²Department of Chemistry, University of California, Berkeley, California 94720, USA

Polymorphism ratio sequencing (PRS) combines the advantages of high-throughput DNA sequencing with new labeling and pooling schemes to produce a powerful assay for sensitive single nucleotide polymorphism (SNP) discovery, rapid genotyping, and accurate, multiplexed allele frequency determination. In the PRS method, dideoxy-terminator extension ladders generated from a sample and reference template are labeled with different energy-transfer fluorescent dyes and coinjected into a separation capillary for comparison of relative signal intensities. We demonstrate the PRS method by screening two human mitochondrial genomes for sequence variations using a microfabricated capillary array electrophoresis device. A titration of multiplexed DNA samples places the limit of minor allele frequency detection at 5%. PRS is a sensitive and robust polymorphism detection method for the analysis of individual or multiplexed samples that is compatible with any four-color fluorescence DNA sequencer.

E-MAIL rich{at}zinc.cchem.berkeley.edu; FAX (510) 642-3599.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.396203.

⁴ Straightforward modifications to the PRS coding/pooling scheme such that the forward and reverse extension reactions are performed simultaneously would reduce the number of reaction tubes by a factor of two. Another factor of two reduction is provided by the use of uniquely-labeled energy transfer (ET)-cassette primers (Berti et al. 2001). For example, by pooling sample and reference templates amplified with uniquely-tailed PCR primers and performing PRS analysis using corresponding cassette-labeled primers, it is feasible to obtain forward and reverse PRS data on both templates with only four reaction tubes—the same number of tubes as dye-terminator sequencing.

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