Optimized Multiplex PCR: Efficiently Closing a Whole-Genome Shotgun Sequencing Project

Genomics. 1999 Dec 15;62(3):500-7. doi: 10.1006/geno.1999.6048.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Algorithms
  • Combinatorial Chemistry Techniques / methods*
  • Evaluation Studies as Topic
  • Genome, Bacterial*
  • Polymerase Chain Reaction / methods*
  • Sequence Analysis, DNA / methods*
  • Streptococcus pneumoniae / genetics