title: The "spanning protocol": a new DNA extraction method for efficient single-cell genetic diagnosis.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  The spanning protocol was most efficient for extracting DNA from a single cell and should be particularly useful for preimplantation genetic diagnosis.
authors.affiliation: Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Tokyo, 160-0016, Japan. incho@tsuchiya-clinic.jp
authors.forename: Shinichi
authors.initials: S
authors.lastname: Tsuchiya
authors.forename: Kou
authors.initials: K
authors.lastname: Sueoka
authors.forename: Noriko
authors.initials: N
authors.lastname: Matsuda
authors.forename: Reiko
authors.initials: R
authors.lastname: Tanigaki
authors.forename: Hironori
authors.initials: H
authors.lastname: Asada
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Hashiba
authors.forename: Shinya
authors.initials: S
authors.lastname: Kato
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Yoshimura
mesh_terms: D001757:Blastomeres / Q000737:chemistry; D020732:Cytogenetic Analysis / Q000379:methods; D004247:DNA / Q000302:isolation & purification*; D005188:False Negative Reactions; D005189:False Positive Reactions; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D019836:Preimplantation Diagnosis / Q000379:methods*
chemical_list: D004247:DNA