[Preliminary exploration of the influence factors on amplification of single cell duplex-nested PCR].
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).


CONCLUSIONS
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.
Zhong
Chang-gao
CG
Institute of Reproduction and Stem Cell Engineering, Central South University; Reproductive & Genetic Hospital, CITIC-XIANGYA, Changsha 410078,China.
Li
Lu-yun
LY
Lu
Chang-fu
CF
Lin
Ge
G
Lu
Guang-xiu
GX
D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D019836:Preimplantation Diagnosis / Q000379:methods*; D019914:Taq Polymerase; D017086:beta-Thalassemia / Q000235:genetics*
D019914:Taq Polymerase