Ostellari L, Bargelloni L, Penzo E, Patarnello P, Patarnello T
Department of Biology, University of Padova, Italy.
Anim Genet. 1996 Dec;27(6):423-7. doi: 10.1111/j.1365-2052.1996.tb00510.x.
We report the isolation and sequencing of 400-550 base pairs (bp) of the mitochondrial DNA (mtDNA) control region of eight species of Sparidae (Perciformes, Teleostei). This sequence information allowed us to design specific primers to one of these species (Pagellus bogaraveo). The new set of primers was used to test a rationalized approach to study the mtDNA nucleotide variability at the intraspecific level. The single-strand conformation polymorphism (SSCP) technique was applied to detect sequence variation in two non-overlapping fragments of the control region of 32 individuals of P. bogaraveo. To assess the sensitivity of the method, the nucleotide sequence of the analysed region was determined for all the specimens. The results showed that, for one of the two fragments, SSCP analysis was able to detect 100% of the underlying genetic variability. In sharp contrast, nucleotide variation of the second DNA fragment was completely unresolved by SSCP under different experimental conditions. This suggests that the resolution power of SSCP is crucially dependent on the nature of the fragment subjected to the analysis; therefore, a preliminary test of the sensitivity of the method should be performed on each specific DNA fragment before starting a large-scale survey. A rationalized approach, combining the SSCP technique and a simplified sequencing procedure, is proposed for studying intraspecific polymorphism at the mtDNA control region in fish.
我们报告了鲷科（鲈形目，硬骨鱼纲）8个物种线粒体DNA（mtDNA）控制区400 - 550碱基对（bp）的分离和测序情况。这些序列信息使我们能够针对其中一个物种（博氏真鲷）设计特异性引物。这套新引物用于测试一种合理的方法，以研究种内水平的mtDNA核苷酸变异性。单链构象多态性（SSCP）技术被应用于检测32个博氏真鲷个体控制区两个不重叠片段中的序列变异。为评估该方法的灵敏度，测定了所有标本分析区域的核苷酸序列。结果表明，对于两个片段中的一个，SSCP分析能够检测到100%的潜在遗传变异。形成鲜明对比的是，在不同实验条件下，SSCP完全无法分辨第二个DNA片段的核苷酸变异。这表明SSCP的分辨能力关键取决于所分析片段的性质；因此，在开始大规模调查之前，应对每个特定的DNA片段进行该方法灵敏度的初步测试。我们提出了一种将SSCP技术与简化测序程序相结合的合理方法，用于研究鱼类mtDNA控制区的种内多态性。
