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

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)407-414
Number of pages8
JournalJournal of Assisted Reproduction and Genetics
Volume22
Issue number11-12
DOIs
Publication statusPublished - 2005 Dec 1

Keywords

  • DNA extraction
  • Nested polymerase chain reaction
  • Preimplantation genetic diagnosis

ASJC Scopus subject areas

  • Reproductive Medicine
  • Genetics
  • Obstetrics and Gynaecology
  • Developmental Biology
  • Genetics(clinical)

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    Tsuchiya, S., Sueoka, K., Matsuda, N., Tanigaki, R., Asada, H., Hashiba, T., Kato, S., & Yoshimura, Y. (2005). The "spanning protocol": A new DNA extraction method for efficient single-cell genetic diagnosis. Journal of Assisted Reproduction and Genetics, 22(11-12), 407-414. https://doi.org/10.1007/s10815-005-7482-x