LoPPS: a long PCR product sequencing method for rapid characterisation of long amplicons

Biochem Biophys Res Commun. 2006 Jun 16;344(4):1080-5. doi: 10.1016/j.bbrc.2006.04.015. Epub 2006 Apr 19.

Abstract

Here, we propose an optimised protocol (LoPPS, long PCR product sequencing) which allows the fast, cost-attractive, and high-throughput sequencing of long PCR products. LoPPS constitutes an alternative to the primer-walking technology which is expensive and time consuming but remains the current standard procedure. It is based on the ultrasonic shearing, polishing, and cloning of PCR or RT-PCR products and is compatible with 96- or 384-well microplate systems in which bacterial growth, preparation of plasmid DNA, and sequencing can be automated. We present results obtained from 24 different RT-PCR products (2.5-4.8 kbp long) obtained from various RNA viruses and fully sequenced using LoPPS. The method proved to be robust and fast. It was successfully used on a low amount of DNA and allowed each target nucleotide position to be controlled twice or more, with a final cost which is one-third of that of primer-walking.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / analysis
  • DNA / chemistry
  • Polymerase Chain Reaction / methods*
  • RNA Viruses / genetics
  • RNA, Viral / analysis
  • RNA, Viral / chemistry
  • Sequence Analysis, DNA / methods*
  • Sequence Analysis, RNA / methods*

Substances

  • RNA, Viral
  • DNA