Cairns M J, Murray V
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia.
Biotechniques. 1994 Nov;17(5):910-4.
We explored the possibility of using a large number of reaction cycles to achieve genomic DNA sequencing in single-copy mammalian genes. A section of the beta-globin promoter was sequenced directly from a sample of human white blood cell DNA. The sequencing fragments were extended from a single, 5'-terminal-labeled oligonucleotide primer by Taq DNA Polymerase in the presence of dideoxyribonucleotides and more than 200 thermal cycles of denaturation, annealing and extension. The labeled sequencing fragments produced in this linear amplification were detected after electrophoresis on a DNA-sequencing gel. We propose that this scheme could be adopted in some instances as an alternative to conventional sequencing.
我们探索了使用大量反应循环来实现单拷贝哺乳动物基因基因组DNA测序的可能性。从人白细胞DNA样本中直接对一段β-珠蛋白启动子进行了测序。在双脱氧核苷酸存在的情况下，由Taq DNA聚合酶从单个5'-末端标记的寡核苷酸引物延伸测序片段，并进行了200多个热循环的变性、退火和延伸。在DNA测序凝胶上进行电泳后，检测在这种线性扩增中产生的标记测序片段。我们提出，在某些情况下，该方案可作为传统测序的替代方法采用。
