A spectrophotometric method to quantify linear DNA.
A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.
Samuel
Manoj
M
Wyeth-Ayerst Research, Collegeville, PA 19355, USA.
Lu
Meiqing
M
Pachuk
Catherine J
CJ
Satishchandran
C
C
D000244:Adenosine Diphosphate / Q000032:analysis / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D004270:DNA, Circular / Q000032:analysis; D003851:Deoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli; D006868:Hydrolysis; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D009243:NAD / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids / Q000032:analysis; D011770:Pyruvate Kinase / Q000378:metabolism; D012343:RNA, Transfer / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods*; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004270:DNA, Circular; D009243:NAD; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase; D003851:Deoxyribonucleases; D008274:Magnesium