title: Development of an ammonium sulfate DNA extraction method for obtaining amplifiable DNA in a small number of cells and its application to clinical specimens.
abstract: DNA extraction from microdissected cells has become essential for handling clinical specimens with advances in molecular pathology. Conventional methods have limitations for extracting amplifiable DNA from specimens containing a small number of cells. We developed an ammonium sulfate DNA extraction method (A) and compared it with two other methods (B and C). DNA quality and quantity, β-globin amplification, and detectability of two cancer associated gene mutations were evaluated. Method A showed the best DNA yield, particularly when the cell number was very low. Amplification of the β-globin gene using DNA from the SNU 790 cell line and papillary thyroid carcinoma (PTC) cells extracted with Method A demonstrated the strongest band. BRAF(V600E) mutation analysis using ethanol-fixed PTC cells from a patient demonstrated both a "T" peak increase and an adjacent "A" peak decrease when 25 and 50 cells were extracted, whereas mutant peaks were too low to be analyzed using the other two methods. EGFR mutation analysis using formalin-fixed paraffin-embedded lung cancer tissues demonstrated a mutant peak with Method A, whereas the mutant peak was undetectable with Methods B or C. Method A yielded the best DNA quantity and quality with outstanding efficiency, particularly when paucicellular specimens were used.
authors.affiliation: Department of Animal Biotechnology, College of Animal Bioscience and Technology, Konkuk University, Gwangjin-gu, Seoul, Republic of Korea.
authors.forename: Seo Young
authors.initials: SY
authors.lastname: Oh
authors.forename: Wook Youn
authors.initials: WY
authors.lastname: Kim
authors.forename: Tae Sook
authors.initials: TS
authors.lastname: Hwang
authors.forename: Hye Seung
authors.initials: HS
authors.lastname: Han
authors.forename: So Dug
authors.initials: SD
authors.lastname: Lim
authors.forename: Wan Seop
authors.initials: WS
authors.lastname: Kim
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry*; D001483:Base Sequence; D002277:Carcinoma / Q000235:genetics; D002291:Carcinoma, Papillary; D002452:Cell Count; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004252:DNA Mutational Analysis; D066246:ErbB Receptors / Q000235:genetics; D005784:Gene Amplification; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000235:genetics; D016707:Tissue Fixation; D055544:beta-Globins / Q000235:genetics
chemical_list: D055544:beta-Globins; D004247:DNA; C512478:EGFR protein, human; D066246:ErbB Receptors; D000645:Ammonium Sulfate