A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.
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.
Wong
K K
KK
Molecular Biosciences Department P7-56, Pacific Northwest National Laboratory, PO Box 999, Richland, WA 99352, USA. kk.wong@pnl.gov
Markillie
L M
LM
Saffer
J D
JD
D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D019793:Fluorescein; D005456:Fluorescent Dyes; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping
D003845:Deoxycytosine Nucleotides; D005456:Fluorescent Dyes; C034817:5-methyldeoxycytidine triphosphate; D004247:DNA; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases; D019793:Fluorescein