中文摘要：

利用焦磷酸测序技术进行核酸序列测定时,测序信号过低或非特异性信号过高均会导致测序失败.因此,PCR引物与测序引物的设计十分重要.本研究以rs8175347为例,通过设计具有不同多聚酶亲和指数（PPI）的PCR引物,并运用焦磷酸测序测定其扩增产物,考察了PPI值对扩增效率以及测序信号的影响；以rs914232、rs671位点为例,对比不同测序方向以及dNTP的推注顺序,考察了两者对测序结果的影响.结果表明,提高PCR引物的PPI值有助于增强扩增效率与测序结果的信号峰强度,测序方向和dNTP推注顺序的不合理选择会严重干扰部分SNP测序结果的判断.因此,在设计PCR引物时,应将多聚酶亲和指数理论与传统基于解链温度值的引物设计思路相结合,为焦磷酸测序提供高质量的测序模板；在进行定量测序时,还要综合考虑待测位点（SNP）两侧的序列,选择合适的测序方向以及dNTP的推注顺序,使测序结果更加准确.

英文摘要：

Pyrosequencing might fail due to a low signal or a high non-specific peak. It is crucial to design polymerase chain reaction （PCR） primers and sequencing primers in order to achieve accurate and reliable pyrosequencing results. By the example of rs8175347, PCR primers with different polymerase preference index （PPI） values were designed, and their amplicons were sequenced to investigate the effects of PPI value on am- plification efficiency and signal intensity; by the examples of rs914232 and rs671, different sequencing direc- tions and dNTP dispensation orders were analyzed to investigate their effects on pyrograms. As a result, a PCR primer with higher PPI value had better amplification efficiency and stronger signal intensity; an inappropriate sequencing direction or dNTP dispensation order decreased the accuracy of genotyping results. Therefore, a new strategy of designing PCR primers was proposed to provide sufficient templates for pyrosequencing by com- bining PPI values with conventional melting temperature-dependent method. Moreover, to obtain accurate quantitative pyrosequencing results, it was suggested to take full consideration of the homogeneous region in the target so as to select an optimum sequencing direction or a dNTP dispensation order. In conclusion, the method proposed here can improve the success rate and quantification accuracy of pyrosequencing.