Abstract
We have developed a procedure for the quantitation of specific DNA which employs nonradioisotopic probes and β-galactosidase as a detector. The sample DNA was immobilized on a nitrocellulose filter paper. After the filter paper had been processed to hybridization with a biotinylated probe DNA, the paper was incubated with avidin-β-galactosidase complex. The optimum ratio of avidin to biotinylated β-galactosidase for preparation of a complex between the two was determined. The filter paper was punched. Each punched piece was put into a microtiter well and β-galactosidase activity was measured using 4-methylumbelliferyl β- d-galactosidase as a substrate. By this method, we were able to quantify as little as a few picograms of specific DNA. The application of this method for the quantitative assay of hepatitis B virus DNA in serum sample is also described. The sensitivity for the detection of the DNA by our method was practically comparable to that of the conventional radioisotopic method. The validity of our method for detection of the virus DNA was further supported by comparison with the serological data.
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