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The "spanning protocol": a new DNA extraction method for efficient single-cell genetic diagnosis.

Shinichi Tsuchiya·Kou Sueoka·Noriko Matsuda·Reiko Tanigaki·Hironori Asada·Tsuyoshi Hashiba·Shinya Kato·Yasunori Yoshimura
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Abstract

PURPOSE: We evaluated methods of preparation of DNA from single cells for amplification and preimplantation genetic diagnosis (PGD), including our "spanning protocol." METHODS: Dystrophin gene exons 45 and 51 were amplified by nested polymerase chain reaction (PCR) from a single lymphocyte or blastomere. Amplification efficiencies were compared between DNA extraction by (A) lysis in distilled water with freeze-thawing and boiling; (B) two-step lysis involving potassium hydroxide and dithiothreitol; and (C) the spanning protocol, using N-lauroylsarcosine. RESULTS: With method A, amplification efficiency was 66/120 (55%) and false-positive such as amplification failure or allele drop out was 42/120 (35%); with B, 96/120 (80%) and 21/120 (17.5%); and with C, 111/120 (92%) and 5/120 (4.2%), using single blastomeres and unaffected lymphocytes from male. Occurrence of false-negative such as contamination of another DNA with method A was 4/120 (3.3%); with B, 10/120 (8.3%); and with C, 2/120 (1.7%) from using single lymphocytes from affected males. CONCLUSION: The spanning protocol was most efficient for extracting DNA from a single cell and should be particularly useful for preimplantation genetic diagnosis.

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BlastomeresCytogenetic AnalysisDNAFalse Negative ReactionsFalse Positive ReactionsFemaleFertilization in VitroHumansLymphocytesMalePolymerase Chain ReactionPreimplantation DiagnosisEvaluation StudyJournal ArticleResearch Support, Non-U.S. Gov't

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Shinichi Tsuchiya, Kou Sueoka, Noriko Matsuda, Reiko Tanigaki, Hironori Asada, Tsuyoshi Hashiba, Shinya Kato, Yasunori Yoshimura. 2005. The "spanning protocol": a new DNA extraction method for efficient single-cell genetic diagnosis.. https://doi.org/10.1007/s10815-005-7482-x

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