Optimized multiplex PCR: Efficiently closing a whole-genome shotgun sequencing project

Hervé Tettelin, Diana Radune, Simon Kasif, Hoda Khouri, Steven L. Salzberg

Research output: Contribution to journalArticlepeer-review

107 Scopus citations


A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory procedures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR primers are used in a single sequencing reaction, is used to interpret the multiplex PCR results. Two protocols are presented, one that minimizes pipetting and another that minimizes the number of reactions. The pipette optimized multiplex PCR method has been employed in the final phases of closing the Streptococcus pneumoniae genome sequence, with excellent results. (C) 1999 Academic Press.

Original languageEnglish (US)
Pages (from-to)500-507
Number of pages8
Issue number3
StatePublished - Dec 15 1999

ASJC Scopus subject areas

  • Genetics


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