The 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.
F. Farzaneh Dehkordi, A.M. Ahadi, A. Shirazi and M. Sadeghizade, 2009. Effect of Lysis Strategy in Accuracy and Repeatability of Sex Determination by Single Cell Polymerase Chain Reaction Method. Journal of Biological Sciences, 9: 78-82.