近交系小鼠双色荧光正相杂交芯片技术体系的建立和优化

目的探讨采用单核苷酸多态性（SNP）检测方法-双色荧光正相杂交芯片技术对近交系小鼠遗传质量监测及相关影响因素。方法运用基于芯片的双色荧光正相杂交检测SNP技术,进行芯片杂交动力学研究,考察信号值（Cy3,Cy5）和ratio值（Cy5/Cy3）与PCR产物点样浓度、PCR产物长度和荧光标记探针长度之间的关系,研究PCR产物点样浓度、PCR产物长度和荧光标记探针长度对SNP分型的影响。结果采用正反标记实验后,Ratio值随着PCR产物点样浓度的增加呈稳定趋势;PCR双链产物长度对信号值影响比较大,点样时其长度不宜太长,最好不超过450 bp;随荧光标记探针长度的增加,基因分型能力明显下降,长度为15 bp最佳,长度超过20 bp时,已基本没有区分能力。结论PCR产物点样浓度、PCR产物长度和荧光标记探针长度是双色荧光正相杂交SNP分型系统的重要影响因素,采取适当的PCR产物点样浓度、PCR产物长度和荧光标记探针长度,并采用正反标记实验,可以取得稳定、准确的基因分型效果。为进一步进行近交系小鼠遗传质量监测的研究奠定基础。

Establishment and Optimization of Dual-Color Fluorescence and Positive Hybridization Chip Technique System in the Inbred Mouse Strains

Objective The aim of this study was to approach the relative factors of using a microarray-based method for scoring a number of genomic DNA in parallel for one SNP marker on a glass slide to serve the genetic monitoring of inbred mice.Methods Hybridization dynamics of the dual-color fluorescence and positive hybridization chip technique were used to optimize the conditions of this method by observing how spotting concentration of PCR products,PCR products length and fluorescence labeled probe length affect genotyping,and the relation between them and the signal value（Cy3,Cy5） and the ratio（Cy5/Cy3）.Results The ratio went stably with the spotting concentration of PCR products keeping increasing by the dye-swap test.The length of PCR products affected signal value notably,which had better be shorter than 450 bp.The longer the fluorescence labeled probe length was,the lower the effect of this SNP genotyping method became.When the length was longer than 20 bp,this technique could not identify the genotype at all.The most suitable length was 15 bp.Conclusion The results of this study indicate that spotting concentration of PCR products,PCR products length and fluorescence labeled probe length are important affecting factors of the dual-color fluorescence and positive hybridization chip technique.With correct spotting concentration and length of PCR products, and correct length of fluorescence labeled probe,this technique is feasible for identifying the inbred mouse strains,which lay the foundation for the genetic monitoring of inbred mice.