Dideoxy genomic sequencing of a single-copy mammalian gene using more than two hundred cycles of linear amplification.
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.
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia.
M J
MJ
Cairns
V
V
Murray
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D015224:Dideoxynucleosides; D005914:Globins / Q000235:genetics*; D006801:Humans; D007962:Leukocytes / Q000737:chemistry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*
D015224:Dideoxynucleosides; D005914:Globins; D004247:DNA