DNA polymerase fluorescent substrates with reversible 3'-tags.
We have synthesized 3'-substituted-2'-deoxyribonucleotide-5'-triphosphates corresponding to A, T, G and C. The 3' position was esterified by a separate anthranylic derivative (3'-tag) giving specific fluorescent properties to each nucleotide (nt). These nt acted as substrates with several DNA polymerases leading to chain termination. Upon alkali or enzymatic treatment of the terminated DNA chain, free 3'-hydroxyl groups were recovered and found able to undergo chain extension when incubated with a mixture of dNTPs and a DNA polymerase. Because each tag has different fluorescent properties in itself, i.e., as a free acid, it theoretically is possible, after removal and characterization of the tag, to infer which nt has been inserted. Reiteration of the process can then be used to determine a nt sequence with a non-gel-based method amenable to automation.
Canard
B
B
Faculté de Médecine, URA-CNRS 1462, Nice, France.
Sarfati
R S
RS
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D001483:Base Sequence; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D013942:Thymine Nucleotides; D062367:ortho-Aminobenzoates / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism
D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D013942:Thymine Nucleotides; D062367:ortho-Aminobenzoates; C066294:3'-anthraniloyl-2'-deoxy-ATP; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate