湿地松、加勒比松改进的SSR-PCR实验方法

Improvement of the SSR-PCR Methodology in PEE and PCH

在开展分子标记辅助湿加松亲本选配研究过程中,为了获得较好的PCR扩增产物,尝试利用了三种不同的PCR实验方法。首先,利用梯度PCR确定不同微卫星引物的最优退火温度,再用降落PCR最大限度地扩增特异产物,最后,当有些反应在电泳中没有检测到产物时,尝试利用巢式PCR再重新扩增,但由于巢式PCR敏感性高,会把特异和非特异的产物放大很多倍,辨别特异谱带时要非常小心。

On the proceeding of a project of Nature Science Foundation of Guangdong, the molecular marker-aided selection of the hybrid parents of Pinus elliottii var. elliottii × Pinus caribaea var. hondurensis, three types of variant PCR methods were attempted to obtain optimally amplified Simple Sequence Repeat products. Gradient PCR was used to determine the optimal annealing temperature （Tm） firstly. Then touchdown PCR was used to amplify the SSR locus effectively. Finally, in the situation where no products detected in the PAGE, nested PCR was carried out to amplify it again using a little product as template DNA. But because of the high sensitivity of the nested PCR, great care must be taken when distinguished the special amplified products.