Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue.
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.
Gilbert
Marcus T P
MT
Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USA. mtpgilbert@gmail.com
Sanchez
Juan J
JJ
Haselkorn
Tamara
T
Jewell
Laurence D
LD
Lucas
Sebastian B
SB
Van Marck
Eric
E
Børsting
Claus
C
Morling
Niels
N
Worobey
Michael
M
D004247:DNA / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D008832:Microchemistry / Q000379:methods; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D016707:Tissue Fixation
D005557:Formaldehyde; D004247:DNA