Hybridization-AT-tailing (HybrAT) method for sensitive and strand-specific detection of DNA and RNA.
delta Tth DNA polymerase catalyzed polymerization of dATP and dTTP into a high-molecular-weight d(A-T) copolymer using oligo-d(A-T) as the template/primer (Hanaki et al., Biochem. Biophys. Res. Commun. 244, 210-219). Taking advantage of this reaction, we developed a highly sensitive method for strand-specific detection of DNA or RNA. The probe consisted of a 40- to 50-base-long complementary sequence on the 5' side and 10 repeats of AT on the 3' side. After hybridization using the 5' side, the 3' side AT repeat region was elongated by delta Tth DNA polymerase in the presence of the dATP, dTTP, and digoxigenin (dig)-11-dUTP. The elongation condition was 52-62 degrees C for 3 h. The method named HybrAT (hybridization-AT-tailing) was at least 100-fold more sensitive than the conventional hybridization with 5' end dig-11-dUTP labeled probe.
Nakajima
N
N
Department of Bacteriology, Faculty of Medicine, University of Tokyo, Japan.
Hanaki
K
K
Shimizu
Y K
YK
Ohnishi
S
S
Gunji
T
T
Nakajima
A
A
Nozaki
C
C
Mizuno
K
K
Odawara
T
T
Yoshikura
H
H
D000225:Adenine; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006526:Hepatitis C / Q000097:blood / Q000175:diagnosis; D018937:Hepatitis C Antibodies / Q000097:blood; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D015345:Oligonucleotide Probes; D011067:Poly dA-dT / Q000737:chemistry*; D012313:RNA / Q000032:analysis*; D012367:RNA, Viral / Q000097:blood; D012680:Sensitivity and Specificity; D013941:Thymine; D014766:Viremia / Q000097:blood
D018937:Hepatitis C Antibodies; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D012367:RNA, Viral; C016023:oligo(dA-dT); D011067:Poly dA-dT; D012313:RNA; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D000225:Adenine; D013941:Thymine