Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase.

Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase.

By incorporating dUMP residues into the 5' end of PCR primers, one can generate products which, after treatment with uracil DNA glycosylase (UDG), contain 3' overhangs. These overhangs can be annealed to vector molecules with complementary overhangs generated in a similar fashion and transformed directly into Escherichia coli without the need for ligase. We have tested this method of ligation-i...

Hetero-stagger cloning: efficient and rapid cloning of PCR products.

A variety of methods have been developed for cloning PCR products, including blunt-end cloning (1), restriction cut back (2), ligation-independent cloning (3), uracil DNA–glycosylase (UDG) treatment of uracil-containing deoxyoligonucleotide primers (4,5) and TA cloning (6–8). Blunt-end cloning of PCR products often requires treatment of PCR products to polish the ends (9). Even with treatments,...

Rapid and reliable cloning of PCR products.

Uracil-DNA glycosylase. Purification and properties of uracil-DNA glycosylase from Micrococcus luteus.

A uracil-DNA-glycosylase from Micrococcus luteus has been purified more than 3,000-fold. The enzyme preparation appears homogeneous, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is devoid of nonspecific endonucleases, specific endonucleases for apurinic and apyrimidinic sites, 3-methyladenine or 7-methylguanine-DNA-glycosylases. It behaves as a monom...

A novel method for site-directed mutagenesis using PCR and uracil DNA glycosylase.

A novel method for site-directed mutagenesis of DNA sequences based on the use of the PCR is described. The method uses two oligonucleotide primers that contain the desired sequence change and overlap at their 5' ends. In addition, the thymine residues in the overlap region have been substituted with deoxyuracil. Amplification of the template plasmid by PCR results in incorporation of the prime...