Padegimas L S, Reichert N A
Department of Plant and Soil Sciences, Mississippi State University, Mississippi State, Mississippi 39762, USA.
Anal Biochem. 1998 Jul 1;260(2):149-53. doi: 10.1006/abio.1998.2719.
An improved method of adaptor ligation PCR was developed for isolation of unknown sequences flanking a known DNA sequence. It was determined that the specificity of the adaptor ligation-based walking technique could be significantly enhanced by using uniquely blocked adaptors along with removal of unligated genomic DNA by exonuclease III digestion. This technique was utilized to isolate three novel promoter regions from three different Zea mays (maize) peroxidase genes. Sequences encoding a putative maize 6-phosphogluconate dehydrogenase gene were also isolated and confirmed by sequence analysis. The described improvements could be applied to other existing adaptor ligation-based PCR walking techniques.
为了分离已知DNA序列侧翼的未知序列，开发了一种改进的衔接子连接PCR方法。结果表明，通过使用独特封闭的衔接子以及用核酸外切酶III消化去除未连接的基因组DNA，可以显著提高基于衔接子连接的步移技术的特异性。该技术被用于从三个不同的玉米过氧化物酶基因中分离出三个新的启动子区域。还分离出了编码假定的玉米6-磷酸葡萄糖酸脱氢酶基因的序列，并通过序列分析得到了证实。所描述的改进可应用于其他现有的基于衔接子连接的PCR步移技术。
