Journal of Biological Sciences1727-30481812-5719Asian Network for Scientific Information10.3923/jbs.2009.78.82DehkordiF. Farzaneh AhadiA.M. ShiraziA. SadeghizadeM. 1200991The main objective of this study was to evaluating of
different methods applicable to the single cell DNA preparation for embryos
sex determination in the early stages. In this study, we have introduced
an efficient protocol for DNA extraction step in the single cell PCR method.
We compared three different lysis methods including; one physical (nitrogen
freezing and thaw) and two chemicals (potassium hydroxide/dithiothreitol;
KOH/DTT lysis protocol) and (Nonidet P40/Proteinase K protocol) for utilizing
in the single cell PCR procedure. The results of multiplex nested single
cell PCR using NP40/PK protocol was eminently more repeatable and efficient
for routine applications in the laboratory in compared with the other
protocols analyzed in this study. Present results showed that the efficiency
of amplification of the target genes in the single blastomer PCR was 94%
with NP40/PK protocol, 88% for KOH/DTT lysis protocol and 64% by using
of freeze and thaw protocol. We concluded that the NP40/PK protocol was
the most efficient method for extracting DNA from a single cell and could
be particularly useful for sexing of bovine embryos in the early stages.]]>Berezovski, M.V., T.W. Mak and S.N. Kryloy,20073879196Bredbackra, P. and O.Y. Finnzymes,2001552334Brown, R.B. and J. Audet,20085131138Chen, C.M., C.L. Hu, C.H. Wang, C.M. Hung, H.K. Wu, K.B. Choo and W.T. Cheng,199954209214Chrenek, P., L. Boulanger, Y. Heyman, P. Uhrin, J. Laurinick, J. Bulla and J.P. Renard,20015510711081Di-Carlo, D., K.H. Jeong and L.P. Lee,20033287291Di-Carlo, D., C. Ionescu-Zanetti, Y. Zhang, P. Hung and L.P. Lee,20055171178Pierce, E.K., J.E. Rice, J.A. Sanchez and J. Lawrence,20023211061111Ray, F.P., M. Vekemans and A. Munnich,20017489494Grifo, J.A., Y.X. Tang and J. Cohen,1992268727729Handyside, A.H., E.H. Kontogianni, K. Hardy and R.M. Winston,1990344768770Hardy, K., R.M.L. Winston and A.H. Handyside,1993in vitro: Failure of cytokinesis during early cleavage.]]>98549558Hasler, J.F., E. Cardey, J.E. Stokes and P. Bradbacka,20025814571469Hussey, N.D., H. Donggue and D.A. Froiland,1999510891094Kontogianni, E.H., D.K. Griffin and A.H. Handyside,199613125132Lee, J.H., J.H. Park, Lee, S.H. Park and D.I. Jim,2004In situ Hybridization (FISH).]]>6214521458Lu, K.Y., A.M. Wo, Y.J. Lo, K.C. Chen, C.M. Lin and C.R. Yang,200622568574Machaty, Z., A. Paldi, T. Csaki, Z. Varga, I. Kiss, Z. Barandi and G. Vajta,199398467470Marc, P.J., C.E. Sims and N.L. Allbritton,20077990549059Park, J.H., J.H. Lee, K.M. Choi, S.Y. Joung and J.Y. Kim et al.,20015518431853Ramalho, M.F.P.D.T., J.M. Garciaa, C.R. Esper, R. Vantini and B.C.A. Alves et al.,20046215691576Schwers, S., A. Ehlich, S. Kobsch, A. Missel and D. Loffert,200031418Thornhill, A.,2007Tsuchiya, S., K. Sueoka, N. Matsuda, R. Tanigaki and H. Asada et al.,200522407411Weikard, R., C. Kuhn, R.M. Brunner, D. Roschlau, C. Pitra, P. Laurent and M. Schwerin,2001601319Zhang, H. and W. Jin,20042510901095Zhang, H. and W. Jin,20061104346351