Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue

Marcus T.P. Gilbert, Juan J. Sanchez, Tamara Haselkorn, Laurence D. Jewell, Sebastian B. Lucas, Eric Van Marck, Claus Børsting, Niels Morling, Michael Worobey

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

Extensive collections of formalin-fixed paraffin-embedded (FFPE) tissues exist that could be exploited for genetic analyses in order to provide important insights into the genetic basis of disease or host/pathogen cointeractions. We report here an evaluation of a 44 SNP multiplex genotyping method, multiplex PCR with minisequencing (MPMS), on 92 DNA extractions performed on six archival FFPE samples of variable DNA quality, which date between 9 and 25 years old. On the three extracts with highest quality, we found the assay efficiency to be near 100%. However, the efficiency of the lowest quality extracts varied significantly. In this study, we demonstrate that although direct measures of DNA concentration in the extracts provide no useful information with regard to subsequent MPMS success, the success of the assay can be determined to some degree a priori, through initial screening of the DNA quality using a simple quantitative real-time PCR (qPCR) assay for nuclear DNA, and/or an assay of the maximum PCR amplifiable size of nuclear DNA. MPMS promises to be of significant use in future genetic studies on FFPE material. It provides a streamlined approach for retrieving a large amount of genetic information using simple, single reactions and minute amounts of archival tissue/DNA. In the light of this evidence, we suggest that the systematic screening of FFPE collections may in the future provide valuable insights into the past.

Original languageEnglish (US)
Pages (from-to)2361-2367
Number of pages7
JournalELECTROPHORESIS
Volume28
Issue number14
DOIs
StatePublished - Jul 1 2007

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Keywords

  • Fixation
  • Formalin
  • Minisequencing
  • Multiplex PCR
  • Tissue

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

Cite this

Gilbert, M. T. P., Sanchez, J. J., Haselkorn, T., Jewell, L. D., Lucas, S. B., Van Marck, E., Børsting, C., Morling, N., & Worobey, M. (2007). Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue. ELECTROPHORESIS, 28(14), 2361-2367. https://doi.org/10.1002/elps.200600589