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BioMed Research International
Volume 2013 (2013), Article ID 546727, 10 pages
http://dx.doi.org/10.1155/2013/546727
Research Article

Development of an Ammonium Sulfate DNA Extraction Method for Obtaining Amplifiable DNA in a Small Number of Cells and Its Application to Clinical Specimens

1Department of Animal Biotechnology, College of Animal Bioscience and Technology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
2Department of Pathology, Konkuk University School of Medicine, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea

Received 4 January 2013; Revised 11 March 2013; Accepted 11 March 2013

Academic Editor: Gjumrakch Aliev

Copyright © 2013 Seo Young Oh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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. BRAFV600E 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.