Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue.
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Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue. / Gilbert, Marcus T P; Sanchez, Juan J; Haselkorn, Tamara; Jewell, Laurence D; Lucas, Sebastian B; Van Marck, Eric; Børsting, Claus; Morling, Niels; Worobey, Michael.
In: Electrophoresis, Vol. 28, No. 14, 2007, p. 2361-7.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue.
AU - Gilbert, Marcus T P
AU - Sanchez, Juan J
AU - Haselkorn, Tamara
AU - Jewell, Laurence D
AU - Lucas, Sebastian B
AU - Van Marck, Eric
AU - Børsting, Claus
AU - Morling, Niels
AU - Worobey, Michael
N1 - Keywords: DNA; Formaldehyde; Humans; Microchemistry; Paraffin Embedding; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; Tissue Fixation
PY - 2007
Y1 - 2007
N2 - 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. Udgivelsesdato: 2007-Jul
AB - 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. Udgivelsesdato: 2007-Jul
U2 - 10.1002/elps.200600589
DO - 10.1002/elps.200600589
M3 - Journal article
C2 - 17578837
VL - 28
SP - 2361
EP - 2367
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 14
ER -
ID: 3848520