Objective: To explore the influence factors on amplification of single cell duplex-nested PCR.
Methods: The mutational loci region CD41-42 and IVS-II 654 of beta-globin gene were amplified by duplex-nested PCR with different combination of primers concentration, different Taq DNA polymerases, different neutralization buffers and with or without predenaturation at 98 degrees C before the PCR amplification in single lymphocyte or single blastomere, thus, to investigate the influence of these factors on the amplification efficiency of PCR.
Results: TaKaRa EX Taq was the most efficient Taq DNA polymerase among different Taq DNA polymerases; primer pair R1+F1 at final concentration of 0.25 micromol/L and R2+F2 at 0.3 micromol/L were the most efficient ones in amplification among different combinations of primers concentrations; the amplification efficiency in neutralization buffer-1 (200 mmol/L Tricine) was obviously higher than that of neutralization buffer-2 (900 mmol/L Tris-HCl, pH 8.3/300 mmol/L KCl/200 mmol/L HCl)(P<0.05); there were no remarkable differences of the amplification efficiency while using whether predenaturation at 98 degrees C before the single cell PCR amplification or not (P>0.05).
Conclusion: There were remarkable differences of the amplification efficiency of single cell duplex-nested PCR while using different combination of primers concentrations, different Taq DNA polymerases, different neutralization buffers. However, predenaturation at 98 degrees C before the single cell PCR amplification could not improve the PCR amplification efficiency.