Terminal phosphate blocked nucleoside polyphosphates
View Patent ↗The present invention describes terminal phosphate blocked nucleoside polyphosphates that are stable at high temperature and their use in nucleic acid amplification and analysis. Current invention further describes charge modified terminal phosphate blocked nucleoside polyphosphates for improved incorporation and direct loading of nucleic acid sequencing reactions onto separating media.
1. In a method for nucleic acid analysis, which includes a high temperature polymerase reaction of a nucleic acid template, a primer, a nucleic acid polymerase, and at least one nucleoside polyphosphate, the improvement comprising conducting said polymerase reaction in the presence of at least one thermally stable terminally blocked nucleoside polyphosphates, wherein said nucleic acid analysis is chain terminator DNA sequencing.
2. The method of claim 1 , wherein said terminally blocked nucleoside polyphosphate is a labeled terminator.
3. The method of claim 1 , wherein said at least one terminally blocked nucleoside polyphosphate has additional negative charges.
4. The method of claim 1 , wherein said at least one terminally blocked nucleoside polyphosphate is net positively charged.
5. The method of claim 1 , wherein said terminally blocked nucleoside polyphosphates are represented by the formula
Z—X—S—B—L
wherein
Z is a terminal block consisting of an organic moiety, including linear or branched, cyclic or acyclic alkyl, alkenyl, alkynyl, aromatic, heterocycle or a detectable label;
X is a polyphosphate chain with at least 3 phosphate or modified phosphate groups;
S is a natural or modified sugar, a carbocycle, or an acyclic linker;
B is a natural or modified heterocyclic base; and
L is H or a linker moiety selected from the group consisting of substituted or unsubstituted, linear or branched cyclic or acyclic alkyl, alkenyl, alkynyl, aromatic, or heterocyclic.
6. The method of claim 5 , wherein at least one of L, B, S, X or Z is substituted with a detectable moiety.
7. The method of claim 5 , wherein at least one of L, B, S, X or Z is substituted with a negatively charged moiety.
8. The method of claim 5 , wherein at least one of L, B, S, X or Z is substituted with a moiety that impart a net positive charge.
9. The method of claim 5 , wherein Z is modified to contain additional negative charges or one or more positive charges.
10. The method of claim 5 , wherein L is labeled with a detectable moiety selected from the group consisting of radioisotopes, electrochemical tags, fluorescent tags, energy transfer (ET) labels, mass spectrometry tags, Raman tags, haptens, chemiluminescent groups, enzymes, chromophores, and any combinations thereof.
11. The method of claim 5 , wherein B is selected from the group consisting of uracil, cytosine, guanine, adenine, thymine, 7-deazaguanine, 7-deazaadenine, hypoxanthine, 7-deazahypoxanthine and 2,6-diaminopurine or derivatives thereof.
12. The method of claim 5 , wherein X is a polyphosphate chain of 3, 4 or 5 phosphate groups.
13. The method of claim 5 for sequencing, wherein S is selected from the group consisting of 2′-deoxyribosyl, 3′-deoxyribosyl, 2, 3′-dideoxyribosyl, 2′, 3′-dideoxy-2′, 3′-didehydroribosyl, 3′-alkoxyribosyl, 3′-azidoribosyl, 3′-aminoribosyl, 3′-fluororibosyl, 3′-mercaptoribosyl, 3′-alkylthioribosyl, 2′-alkoxyribosyl, 2′-azidoribosyl, 2′-aminoribosyl, 2′-fluororibosyl, 2′-mercaptoribosyl, 2′-alkylthioribosyl, carbocyclic, acyclic and other modified sugars.
14. The method of claim 5 , wherein said terminally blocked nucleoside polyphosphate is represented by the formula
Where n is 1,2or 3;
Block is an organic moiety or H when X is CH 2 ;
X is CH 2, O, S or NH;
Y is O, H or BH 3 ;
R is H or OH;
Base is a natural or modified nucleoside base;
Linker is H, alkyl, aryl, alkylaryl, alkenyl, alkynyl, aromatic, or heterocyclic and may optionally be charged; and
Z is a fluorescent dye, an energy transfer dye, a chemiluminescent compound, a colored compound, a mass tag or no label at all.