Preparation of single-stranded deoxyribonucleic acid probes using an immobilized template.
A new method for the easy preparation of specific single-stranded DNA fragments is presented. Recombinant M13 DNA containing the strand complementary to the sequence of interest is made partially double-stranded by elongating a conventional M13 sequencing primer. Following linearization by enzymatic digestion downstream from the insert (relative to priming site), this DNA is coupled to diazotized paper through its single-stranded (vector) portion. Subsequent denaturation of the double-stranded region generates an immobilized template strand. Successive runs of primed syntheses of the (desired) complementary strand can be realized using the same template. The copies are easily isolated by release upon denaturation. DNA probes prepared by this method have proven to be valuable tools for gene analysis.
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D013698:Templates, Genetic
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded