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Nucleic Acids Research, 2002, Vol. 30, No. 7 e31
© 2002 Oxford University Press

Multiplex Pyrosequencing

Nader Pourmand1, Elahe Elahi1,2, Ronald W. Davis1 and Mostafa Ronaghi1,*

1Stanford Genome Technology Center, Stanford University, 855 California Avenue, Palo Alto, CA 94304, USA and 2Faculty of Science, Tehran University, Tehran, Iran

We describe here the development of a new and simple single-tube multiplex Pyrosequencing assay. Genomic DNA or cDNA was employed to PCR amplify region(s) using biotinylated and normal primer(s). Subsequent to capture of PCR products on streptavidin-coated beads, single-stranded DNA separation and hybridization of multiple sequencing primers, Pyrosequencing was performed. The obtained pyrogram resulted in a unique pattern in which the intensity of the signal determined the number of incorporated nucleotide(s). Here, we demonstrate the use of this multiplex Pyrosequencing for single nucleotide polymorphisms genotyping and microbial typing.

* To whom correspondence should be addressed. Tel: +1 650 812 1971; Fax: +1 650 812 1975; Email: mostafa{at}stanford.edu


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