Nonradioactive labeling with chemically modified cytosine tails by the polymerase chain reaction.
We developed a modified nonradioactive method for the detection of DNA. This method makes use of the polymerase chain reaction for preparation of probes; that is, a DNA fragment inserted in the polylinker region of an M13 or pUC vector is amplified with primers that have a modified cytosine tail at the 5' terminus (C-tailed primers). By this method, large amounts of labeled probes can be obtained easily. After hybridization, modified cytosine tails can be detected immunologically. DNA labeled by this method could be used in plaque hybridization. We could detect 0.05 pg of dot-blotted labeled DNA in 30 min with an enzyme-catalyzed chemiluminescence reaction.
Uchimura
Y
Y
Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Shiga, Japan.
Ishida
H
H
Asada
K
K
Mukai
H
H
Kato
I
I
D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives*; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D007118:Immunoassay / Q000379:methods*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011066:Poly C / Q000378:metabolism; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D009838:Oligodeoxyribonucleotides; C023826:poly(dC); D011066:Poly C; D003596:Cytosine; D004247:DNA