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首页> 外文期刊>Journal of the American Society for Horticultural Science >PCR-based single-strand conformation polymorphism (SSCP) analysis to clone nine aquaporin genes in cucumber
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PCR-based single-strand conformation polymorphism (SSCP) analysis to clone nine aquaporin genes in cucumber

机译：基于PCR的单链构象多态性（SSCP）分析可克隆黄瓜中的9个水通道蛋白基因

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摘要

Combining the use of PCR and single-strand conformation polymorphisms (SSCP), nine sequences from the cucumber genome were successfully identified and cloned that encoded two well-conserved asparagine-proline-alanine (NPA) domain homologues to aquaporin genes. The sensitivity and detection efficiency of SSCP and restriction enzyme analysis for detecting DNA sequence variation were evaluated using similar-sized DNA fragments. The SSCP analysis was more sensitive and efficient for discriminating different clones than restriction enzyme analysis, although some sequence variation inside similar-sized DNA fragments could be identified by restriction analysis. Consideration of the results of SSCP analysis with DNA sequence information indicated that one or two base pair changes in the amplified regions could be detected. Moreover, the SSCP analysis results of genomic DNA PCR products that were amplified by degenerate primers can provide rough information about the number of member genes. If the SSCP bands of a cloned fragment (such as CRB7) did not have the corresponding bands from genomic DNA PCR products, that fragment might be a misamplified product. The PCR-based SSCP method with degenerate oligonucleoticle primers should facilitate the cloning of member genes.
机译：结合使用PCR和单链构象多态性（SSCP），从黄瓜基因组中成功鉴定并克隆了9个序列，该序列编码了两个与水通道蛋白基因高度保守的天冬酰胺-脯氨酸-丙氨酸（NPA）域同源物。使用相似大小的DNA片段评估了SSCP的敏感性和检测效率以及限制酶分析法检测DNA序列变异的能力。尽管可以通过限制性酶切分析识别相似大小的DNA片段内部的一些序列变异，但SSCP分析对于区分不同克隆比灵敏和高效。考虑到具有DNA序列信息的SSCP分析的结果表明，可以检测到扩增区域中的一个或两个碱基对变化。此外，通过简并引物扩增的基因组DNA PCR产物的SSCP分析结果可提供有关成员基因数量的粗略信息。如果克隆的片段（例如CRB7）的SSCP条带没有基因组DNA PCR产物的相应条带，则该片段可能是扩增错误的产物。基于PCR的简并寡核苷酸引物的SSCP方法应有助于克隆成员基因。

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