The “Spanning Protocol”: A new DNA extraction method for efficient single-cell genetic diagnosis
S Tsuchiya, K Sueoka, N Matsuda, R Tanigaki… - Journal of Assisted …, 2005 - Springer
S Tsuchiya, K Sueoka, N Matsuda, R Tanigaki, H Asada, T Hashiba, S Kato, Y Yoshimura
Journal of Assisted Reproduction and Genetics, 2005•SpringerPurpose: 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 …
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 …
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.
Springer