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Extraction of DNA from human embryos after long‐term preservation in formalin and B ouin's solutions
Author(s) -
Nagai Momoko,
Minegishi Katsura,
Komada Munekazu,
Tsuchiya Maiko,
Kameda Tomomi,
Yamada Shigehito
Publication year - 2016
Publication title -
congenital anomalies
Language(s) - English
Resource type - Journals
eISSN - 1741-4520
pISSN - 0914-3505
DOI - 10.1111/cga.12148
Subject(s) - term (time) , extraction (chemistry) , dna extraction , chemistry , dna , embryo , chromatography , biology , microbiology and biotechnology , biochemistry , gene , physics , polymerase chain reaction , quantum mechanics
Abstract The “ K yoto C ollection of H uman E mbryos” at K yoto U niversity was begun in 1961. Although morphological analyses of samples in the K yoto C ollection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or B ouin's solution for 20–50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long‐term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long‐term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long‐term fixation, including DNA ‐protein crosslinking damage. Diluting Li 2 CO 3 with 70% ethanol effectively removed picric acid from samples fixed in B ouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long‐term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295‐ and 838‐bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the K yoto C ollection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies.