A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.
AUTOR(ES)
Wong, K K
RESUMO
Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=147012Documentos Relacionados
- PCR with 5-methyl-dCTP replacing dCTP.
- An improved method for partial restriction digestion of ultraviolet irradiated DNA.
- A simple method for sequencing small DNAs by introducing precise overlapping ends into restriction digestion fragments.
- A simple method for sequencing small DNAs by inducing precise overlapping ends into restriction digestion fragments.
- A method for sequencing restriction fragments with dideoxynucleoside triphosphates.