单细胞二重巢式PCR扩增影响因素初探

目的: 对影响单细胞二重巢式PCR扩增的因素进行初步探讨.方法:针对β-珠蛋白基因CD41-42、IVS-Ⅱ654突变位点区域采用二重巢式PCR,分别以不同的引物浓度组合、不同的Taq DNA聚合酶、不同的中和缓冲液、PCR反应前采用或不采用98 ℃预变性扩增单个淋巴细胞和/或单卵裂球,了解这些因素对PCR扩增效率的影响.结果: 不同的引物浓度组中,终浓度为0.25 μmol/L R1 F1引物对与 0.3 μmol/L R2 F2引物对配对扩增效率最高;不同的Taq DNA聚合酶中TaKaRa EX Taq的扩增效率最高;中和缓冲液-1(200 mmol/L Tricine)的扩增效率显著高于中和缓冲液-2(900 mmol/L Tris·HCl, pH 8.3/300 mmol/L KCl/200 mmol/L HCl)的扩增效率(P<0.05);单细胞PCR反应前采用98 ℃预变性与不采用98 ℃预变性的扩增效率间差异无统计学意义.结论:不同引物浓度组合、不同的Taq DNA聚合酶、不同的中和缓冲液对单细胞的二重巢式PCR的扩增效率存在明显差异,而PCR反应前采用98 ℃预变性不能提高PCR扩增效率.

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). 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.