The "spanning protocol": a new DNA extraction method for efficient single-cell genetic diagnosis.
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.
Tsuchiya
Shinichi
S
Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Tokyo, 160-0016, Japan. incho@tsuchiya-clinic.jp
Sueoka
Kou
K
Matsuda
Noriko
N
Tanigaki
Reiko
R
Asada
Hironori
H
Hashiba
Tsuyoshi
T
Kato
Shinya
S
Yoshimura
Yasunori
Y
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*
D004247:DNA