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 language | English |
|---|---|
| Pages (from-to) | 407-414 |
| Number of pages | 8 |
| Journal | Journal of Assisted Reproduction and Genetics |
| Volume | 22 |
| Issue number | 11-12 |
| DOIs | |
| Publication status | Published - 2005 Dec |
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Keywords
- DNA extraction
- Nested polymerase chain reaction
- Preimplantation genetic diagnosis
ASJC Scopus subject areas
- Obstetrics and Gynaecology
- Developmental Biology
- Genetics
- Reproductive Medicine
Cite this
The "spanning protocol" : A new DNA extraction method for efficient single-cell genetic diagnosis. / Tsuchiya, Shinichi; Sueoka, Kou; Matsuda, Noriko; Tanigaki, Reiko; Asada, Hironori; Hashiba, Tsuyoshi; Kato, Shinya; Yoshimura, Yasunori.
In: Journal of Assisted Reproduction and Genetics, Vol. 22, No. 11-12, 12.2005, p. 407-414.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - The "spanning protocol"
T2 - A new DNA extraction method for efficient single-cell genetic diagnosis
AU - Tsuchiya, Shinichi
AU - Sueoka, Kou
AU - Matsuda, Noriko
AU - Tanigaki, Reiko
AU - Asada, Hironori
AU - Hashiba, Tsuyoshi
AU - Kato, Shinya
AU - Yoshimura, Yasunori
PY - 2005/12
Y1 - 2005/12
N2 - 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.
AB - 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.
KW - DNA extraction
KW - Nested polymerase chain reaction
KW - Preimplantation genetic diagnosis
UR - http://www.scopus.com/inward/record.url?scp=28744447279&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=28744447279&partnerID=8YFLogxK
U2 - 10.1007/s10815-005-7482-x
DO - 10.1007/s10815-005-7482-x
M3 - Article
C2 - 16331538
AN - SCOPUS:28744447279
VL - 22
SP - 407
EP - 414
JO - Journal of Assisted Reproduction and Genetics
JF - Journal of Assisted Reproduction and Genetics
SN - 1058-0468
IS - 11-12
ER -