Buĭdakov V M, Aslaev A N, Gumerov A A, Tuĭgunov M M, Lukmanova G I
Med Parazitol (Mosk). 2010 Jul-Sep(3):3-5.
This study was undertaken to analyze the nucleotide sequences of a marker fragment in the mitochondrial cox1 gene in polymorphic variants of G1 strain from the nucleotide sequence bank "Genbank" and to choose conditions for a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to differentiate the G1 genotype in E.granulosus isolates. The analysis indicated that G1 genotype polymorphism was due to impact nucleotide replacements and to the varying length of the marker fragment of the coxl gene (by the presence of absence of the 5' GTGGCT 3' site with the coordinates 10275-10280). The procedure of PCR-RFLP was modified to identify the G1 variants due to the varying coxl length. New primers annealed to the variable coxl site of the following structure: 5' TGTGTTGATTTT-GCCTGG 3' (direct); 5' GCCACCACAAACCAAGTATC 3' (inverse) were chosen. Then the localizations of restriction sites were determined for the endonucleases R.Fok 1, R.Sfa NI, and R.Mael and the restriction fragment length was calculated for the RFLP analysis.
本研究旨在分析来自核苷酸序列库“Genbank”的G1株多态性变体中线粒体cox1基因标记片段的核苷酸序列,并选择聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法的条件,以区分细粒棘球绦虫分离株中的G1基因型。分析表明,G1基因型多态性是由于影响核苷酸替换以及cox1基因标记片段长度的变化(取决于坐标为10275-10280的5' GTGGCT 3'位点的有无)。由于cox1长度不同,对PCR-RFLP程序进行了修改以鉴定G1变体。选择了与以下结构的可变cox1位点退火的新引物:5' TGTGTTGATTTT-GCCTGG 3'(正向);5' GCCACCACAAACCAAGTATC 3'(反向)。然后确定了限制性内切酶R.Fok 1、R.Sfa NI和R.Mael的限制性位点定位,并计算了用于RFLP分析的限制性片段长度。