Comparison of the DNA extraction methods for polymerase chain reaction amplification from formalin-fixed and paraffin-embedded tissues.
To obtain an adequate quality and quantity of DNA from formalin-fixed and paraffin-embedded tissue, six different DNA extraction methods were compared. Four methods used deparaffinization by xylene followed by proteinase K digestion and phenol-chloroform extraction. The temperature of the different steps was changed to obtain higher yields and improved quality of extracted DNA. The remaining two methods used microwave heating for deparaffinization. The best DNA extraction method consisted of deparaffinization by microwave irradiation, protein digestion with proteinase K at 48 degrees C overnight, and no further purification steps. By this method, the highest DNA yield was obtained and the amplification of a 989-base pair beta-globin gene fragment was achieved. Furthermore, DNA extracted by means of this procedure from five gastric carcinomas was successfully used for single strand conformation polymorphism and direct sequencing assays of the beta-catenin gene. Because the microwave-based DNA extraction method presented here is simple, has a lower contamination risk, and results in a higher yield of DNA compared with the ordinary organic chemical reagent-based extraction method, it is considered applicable to various clinical and basic fields.
Sato
Y
Y
Department of Molecular Diagnostics, School of Allied Health Sciences Kitasato University, Kanagawa, Japan. yuichi@med.kitasato-u.ac.jp
Sugie
R
R
Tsuchiya
B
B
Kameya
T
T
Natori
M
M
Mukai
K
K
D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D005557:Formaldehyde; D005784:Gene Amplification; D006801:Humans; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D016707:Tissue Fixation / Q000379:methods
D017931:DNA Primers; D004273:DNA, Neoplasm; D005557:Formaldehyde