Oligonucleotide tagged nucleoside triphosphates (OTNTPs) for genetic analysis
View Patent ↗Oligonucleotide tagged nucleoside triphosphates, OTNTPs, which are substrates for polymerases and or terminal nucleotidyl transferases are provided as well as methods of making these OTNTPs. Further provided are OTNTPs with fluorescent dyes including energy transfer dyes, attached to the oligonucleotide chain, OTNTPs with unnatural bases incorporated in the oligonucleotide sequence and methods for incorporating these OTNTPs in DNA or RNA. Also provided are methods for using the oligonucleotides on OTNTPs for amplifying the oligo sequence on the OTNTP using an amplification method described above. Further provided are reactive bifunctional amidites, methods of making these compounds and methods for detecting single nucleotide polymorphisms using the above OTNTPs. Methods for detecting differential gene expression using the OTNTPs and methods of separating specifically modified DNA or RNA using the OTNTPs are also provided.
1. An OTNTP comprising the structure
wherein
R and R′ independently represent H, OH, OCH 3 , CH 3 , N 3 , SH, NCO, NCS, SCN NH 2 , F, Cl or NR″R′″,
wherein R″ and R′″ independently represent H or a small chain C1–C5 alkyl;
X represents O, S, CH 3 or BH 3 ;
Base represents uracil, cytosine, thymine, adenine, guanine, hypoxanthine, 2-aminopurine, 2,6-diaminopurine, xanthene, deazaadenine, deazaguanine or an analog thereof;
Linker represents a linear or branched alkynyl, alkenyl, alkyl, alkylaryl, aryl, or arylalkyl containing one or more heteroatoms selected from the groups consisting of N, O, S and P, and is attached to the base;
N represents a ribose, deoxyribose, a natural or unnatural deoxyribonucleoside or a natural or unnatural ribonucleoside, wherein one or more of the N's is labeled with a fluorescent dye;
n represents an integer from 10 to 100; and
wherein each N is linked to at least one other N by phosphodiester, phosphorothioate, alkyl phosphonate, or other linkages.
2. An OTNTP comprising the structure
wherein
R and R′ independently represent H, OH, OCH 3 , CH 3 , N 3 , SH, NCO, NCS, SCN NH 2 , F, Cl or NR″R′″,
wherein R″ and R′″ independently represent H or a small chain C1–C5 alkyl
X represents O, S, CH 3 or BH 3 ;
Base represents uracil, cytosine, thymine, adenine, guanine, hypoxanthine, 2-aminopurine, 2,6-diaminopurine, xanthene, deazaadenine, deazaguanine or an analog thereof;
Linker represents a linear or branched alkynyl, alkenyl, alkyl, alkylaryl, aryl, or arylalkyl containing one or more heteroatoms selected from the groups consisting of N, O, S and P, and is attached to the base;
N represents a ribose, deoxyribose, a natural or unnatural deoxyribonucleoside or a natural or unnatural ribonucleoside, wherein one of the N's is coupled to a donor dye and another N is coupled to an acceptor dye and there is energy transfer between the donor and acceptor when the donor dye is excited;
n represents an integer from 10 to 100; and
wherein each N is linked to at least one other N by phosphodiester, phosphorothioate, alkyl phosphonate, or other linkages.
3. An OTNTP comprising the structure
wherein
R and R″are both H;
X represents O, S, CH 3 or BH 3 ;
Base represents uracil, cytosine, thymine, adenine, guanine, hypoxanthine, 2-aminopurine, 2,6-diaminopurine, xanthene, deazaadenine, deazaguanine or an analog thereof;
Linker represents a linear or branched alkynyl, alkenyl, alkyl, alkylaryl, aryl, or arylalkyl containing one or more heteroatoms selected from the groups consisting of N, O, S and P, and is attached to the base;
N represents a ribose, deoxyribose, a natural or unnatural deoxyribonucleoside or a natural or unnatural ribonucleoside,
optionally containing a detectable moiety;
n represents an integer from 10 to 100; and
wherein each N is linked to at least one other N by phosphodiester, phosphorothioate, alkyl phosphonate, or other linkages.