Comparison Of Different Lysis Methods In The Single Cell Analysis Of The Efficiency And Allele Dropout Rate Of Polymerase Chain Reaction For Preimplantation Genetic Diagnosis

Objective To assess the amplification efficiency and diagnosis accuracy andallele dropout(ADO) rates of single cell nested polymerase chain reaction(PCR) bycomparing four different lysis methods ;and to search the best lysis method in aneffort both to maximize PCR amplification and diagnosis accuracy at the sametime minimize ADO using amelogenin gene; and to apply the best lysis method tothe analysis of single pre-embryo blastomeres.Methods 400 male and 200 famale single lymphocytes were collected anddivided randomly into four groups (n=150/group) and prepared under the fourfollowing conditions: (1) water only, freeze-thaw liquid nitrogen then boiling; (2)potassium hydroxide/dithiotheriol (KOH/DTT), heated to650C,followed by acidneutralization buffer; (3) proteinase K/sodium dodecyl sulphate (PK/SDS),heated to 37℃for 1h,followed by boiling the mixture at 98℃for 15min;(4) proteinase K /Tween-20(PK/Tween-20), heated to 55℃for 3h,followedby boiling the mixture at 98℃for 15min; 20 single blastomeres were preparedunder PK/Tween-20. Then cells were analyzed by PCR using nested primersamplification with amelogenin gene. In every PCR, positive and negative controlwere settled.Results The amplification efficiencies of the single lymphocytes in water only,freeze-thaw liquid nitrogen then boiling, KOH/DTT , PK/SDS , PK/Tween-20were 61.33%,86%,90%,95.33%, respectively ;the diagnosis accuracy were76.08%,93.02%,89.26%,96.5%, respectively ;the allele dropout rate for malelymphocytes were 53.85%,29.55%, 29.35%, 12.5%, respectively. Theamplification efficiencies and diagnosis accuracy of the latter three methods aremuch higher than the first method, but the ADO are much fewer (P<0.005).The amplification efficiencies and diagnosis accuracy among the latter threemethods are without statistical meaning (P>0.05 ).The ADO between secondand third method are without statistical meaning two (P>0.05 ).The fourthmethod has the lowest ADO rate of all (P<0.005) .The single blastomere haslower efficiency than single lymphocyte (P<0.05).Conclusion The PK/Tween-20 for DNA preparation is superior to the othermethods evaluated .