Restriction enzyme cleavage of fluorescently labeled DNA fragments--analysis of the method and its usage in examination of digestion completeness.
Restriction enzymes have proven to be among the most valuable tools in molecular biology. In this work, we demonstrate that the cleavage of fluorescently labeled, PCR-amplified DNA can be used as a simple and highly sensitive technique for detection of sequences present in a percentage as low as 0.6% in a DNA pool. Due to the fact that fluorescent labeling of DNA fragments enables such sensitive detection and quantification of restriction enzyme cleavage, the method was further exploited in monitoring of the enzymatic digestion completeness and in determination of factors that influence restriction enzyme effectiveness. We analyzed the activity of six restriction endonucleases; the percentage of uncleaved DNA fragments predominantly ranged between 2.0 and 2.5 and the highest value was 8.00%. We conclude that, since the enzymatic digestion completeness may not always be assured, each assay based on restriction enzyme cleavage that is intended to be used in investigations of heterogeneity in a DNA pool should be constructed so that the presence of cleaved sequences is the indication of pool nonuniformity. When the presence of uncleaved sequences indicates pool heterogeneity, the results could be misleading due to possible incompleteness of enzymatic cleavage.
Ivancic-Jelecki
Jelena
J
Molecular Biomedicine Unit, Department of Research and Development, Institute of Immunology Inc., Rockefeller Street 10, 10000 Zagreb, Croatia. jivancic@imz.hr
Baricevic
Marijana
M
Santak
Maja
M
Forcic
Dubravko
D
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006868:Hydrolysis; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA; D004262:DNA Restriction Enzymes