Preparation of sensitive and specific oligonucleotide probes tailed using terminal transferase and dITP.
An oligonucleotide probe tailed with deoxyadenosine-5'-triphosphate or deoxythymine-5'-triphosphate is detectable with high sensitivity, but has a major drawback--the tail co-hybridizes specifically to complementary sequences. This can be a problem when screening cDNA clones that contain poly(dA) sequences. While it is possible to mask the cDNA tail with unlabeled poly(dA) or poly(A) oligonucleotides, false-positive clones are still produced because complete masking of extremely long (dA) tails is difficult. As a result, only cDNA clones that have extremely long poly(dA) sequences are often obtained by hybridization screening using tailed probes. In this report, we describe an oligonucleotide probe tailed with DIG-labeled nucleotide in combination with deoxyinosine-5'-triphosphate that was highly specific and sensitive to cDNAs. Terminal deoxynucleotidyl transferase efficiently adds dI nucleotides to the 3'-end. The dI of the tails did not pair with any nucleotides under stringent hybridization so that the specificity of hybridization assays remained high without affecting the sensitivity of the test. Colony hybridization experiments demonstrated that there were very few (1 of 80 tested) false positives using this technique. Its use may increase the accuracy of cDNA screening.
Sugiyama
T
T
Helix Research Institute, Chiba, Japan. sugiyama@hri.co.jp
Ishii
S
S
Saito
K
K
Yamamoto
J
J
Isogai
T
T
Ota
T
T
D004253:DNA Nucleotidylexotransferase / Q000494:pharmacology*; D003838:Deoxyadenine Nucleotides; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity
D003838:Deoxyadenine Nucleotides; D015345:Oligonucleotide Probes; D004253:DNA Nucleotidylexotransferase; C026600:2'-deoxyadenosine triphosphate