The profiling of pre- and post-warming DNA in mouse embryos with microsatellite method

Abstract

Aim: This research aimed to identify the deoxyribonucleic acid (DNA) profile and changes of post-warming embryo after being frozen with vitrification method using microsatellite method. Materials and Methods: This research examined the mouse embryo blastocysts that were divided into four groups: Post-warming living blastocyst, post-warming living blastocyst with half fragmented cell, post-warming dead blastocyst, and pre-freezing living blastocyst. The isolation sample applied phenol-chloroform method. After obtaining polymerase chain reaction results, all the samples of pre-freezing fresh embryo, post-warming living embryo, dead embryo, and degenerated embryo were then examined by single-strand conformation polymorphism (SSCP). Results: The amplification with D18mit14 primer was 100 bp and 150bp with D18mit87 primer, 150bp with D7mit22, and 300bp with D7mit25. The result of SSCP with D18mit14 primer showed that the blastocysts were fragmented and dead after warming process and formed into two DNA strand fragments, while the fresh embryos which passed freezing process did not form any fragment. D18mit87 primer SSCP indicated different fragments for each treatment. The result of SSCP using D7mit22 formed two different fragments for each treatment. While using D7mit25, the SSCP result formed some different fragments for each sample. Post-warming living embryo had similar ribbon to pre-freezing fresh embryo. Conclusion: D7mit222, D7mit25, and D18mit87 primers could be used as the aneuploidy marker on mouse embryos that were induced by post-warming process. The profile of living blastocyst, dead blastocyst, and post-warming fragmented blastocyst had different DNA tapes.

Keywords

• blastocyst
• DNA mutation
• single-strand conformation polymorphism
• vitrification