Functional assessment for elimination of mismatches in nuclear and whole cell extracts obtained from mouse and human blastocysts.
Preimplantation embryos may have an increased risk of having mismatches due to the rates of cell proliferation and DNA replication. Elimination of mismatches in human gametes and embryos has not been investigated. In this study we developed a sensitive functional assay to examine the repair or elimination of mismatches in both commercially available cell extracts and extracts obtained from preimplantation embryos. Heteroduplex molecules were constructed using synthetic oligonucleotides. Efficiency of the repair of mismatches was semi-quantitatively analysed by exposure to nuclear/whole cell extracts (as little as 2.5 µg) and extracts obtained from pooled mouse and human blastocysts to investigate the repair capacity in human embryos. A cell free in vitro assay was successfully developed to analyze the repair of mismatches using heteroduplex complexes. The assay was further optimized to analyze repair of mismatches in cell extracts obtained from oocytes and blastocysts using minute amounts of protein. The efficiency of mismatch repair was examined in both mouse and human blastocysts (2.5 µg). The blastocysts were observed to have a lower repair efficiency compared to commercially available nuclear and whole cell extracts. In conclusion, a sensitive, easy, and fast in vitro technique was developed to detect the repair of mismatch efficiency in embryos.
a Department of Medical Genetics , Faculty of Medicine, Near East University , Nicosia , Cyprus.|b Preimplantation Genetics Group, Institute for Women's Health , University College London , London , UK.
Pinar
P
Tulay
b Preimplantation Genetics Group, Institute for Women's Health , University College London , London , UK.
Souraya
S
Jaroudi
c The Centre for Reproductive and Genetic Health , University College Hospital , London , UK.
Alpesh
A
Doshi
b Preimplantation Genetics Group, Institute for Women's Health , University College London , London , UK.
Sioban B
SB
SenGupta
D000818:Animals; D020137:Base Pair Mismatch; D001755:Blastocyst / Q000378:metabolism*; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism*; D004260:DNA Repair; D006801:Humans; D051379:Mice
D002457:Cell Extracts
DNA repair; elimination of mismatches; embryo; functional assay; human