Derivatives utilizable in nucleic acid sequencing
First Claim
Patent Images
1. An ester of a deoxyribonucleoside or ribonucleoside 5'"'"' triphosphate having formula (II):
- ##STR8## where X is a fluorophore or hydrogen, in which the 3'"'"' hydroxyl can be restored by cleavage of the caproic acid when substituted by said fluorophore in basic medium or by action of an enzyme.
0 Assignments
0 Petitions
Accused Products
Abstract
Deoxyribonucleotide 5'"'"' triphosphate (dNTP) or ribonucleotide 5'"'"' triphosphate (NTP) esters for use in a nucleic acid sequencing process without use of a gel and having one o formulae (I), (II), (III) or (IV).
-
Citations
19 Claims
-
1. An ester of a deoxyribonucleoside or ribonucleoside 5'"'"' triphosphate having formula (II):
- ##STR8## where X is a fluorophore or hydrogen, in which the 3'"'"' hydroxyl can be restored by cleavage of the caproic acid when substituted by said fluorophore in basic medium or by action of an enzyme.
- View Dependent Claims (2)
-
3. An ester of a deoxyribonucleoside or ribonucleoside 5'"'"' triphosphate of formula (III) or (IV):
- ##STR9## where X is a fluorophore or hydrogen, and in which the 3'"'"' hydroxyl group can be liberated in two steps which are;
oxidation of the vicinal diol by periodate; and enolization and β
-elimination of the 3'"'"' aldehyde when said ester has formula (III), orelimination as a result of the action of hydrazine when said ester has formula (IV).
- ##STR9## where X is a fluorophore or hydrogen, and in which the 3'"'"' hydroxyl group can be liberated in two steps which are;
-
4. A method of sequencing a polynucleotide chain, comprising the steps of:
-
(a) constructing a primer; (b) adding to said primer by a nucleic acid polymerase an ester of a deoxyribonucleoside or ribonucleoside 5'"'"' triphosphate of formula (II), (III) or (IV) esterified at the 3'"'"' OH such that its incorporation thereof blocks any subsequent elongation and said ester bears a label specific for each of the four bases of said esterified nucleotides; (c) hydrolyzing the 3'"'"' OH function by chemical or enzymatic hydrolysis; (d) characterizing the hydrozylate formed for a given nucleotide; (e) repeating steps (a)-(d) in order to characterize the next nucleotide, ##STR10## where X is a fluorophore or hydrogen, in which the 3'"'"' hydroxyl can be restored by cleavage of the caproic acid when substituted by said fluorophore in basic medium or by action of an enzyme;
##STR11## where X is a fluorophore or hydrogen, and in which the 3'"'"' hydroxyl group can be liberated in two steps which are;oxidation of the vicinal diol by periodate; and enolization and β
-elimination of the 3'"'"' aldehyde;
##STR12## where X is a fluorophore or hydrogen, and in which the 3'"'"' hydroxyl group can be liberated in two steps which are;oxidation of the vicinal diol by periodate; and elimination as a result of the action of hydrazine. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12)
-
- 13. The method of claim 13, wherein said nucleic acid polymerase is Taq polymerase.
-
17. A kit for the diagnosis of the presence in a sample of nucleic acid sequence, comprising:
-
(a) a sequencing primer; (b) four esters of a deoxyribonucleoside or ribonucleoside 5'"'"' triphosphate of formula (II), (III) or (IV); and (c) a nucleic acid polymerase which lacks a 3'"'"'-5'"'"' exonucleolytic activity; and (d) optionally, a solid phase to immobilize the nucleic acid to be analyzed or said primer, ##STR13## where X is a fluorophore or hydrogen, in which the 3'"'"' hydroxyl can be restored by cleavage of the caproic acid when substituted by said fluorophore in basic medium or by action of an enzyme;
##STR14## where X is a fluorophore or hydrogen, and in which the 3'"'"' hydroxyl group can be liberated in two steps which are;oxidation of the vicinal diol by periodate; and enolization and β
-elimination of the 3'"'"' aldehyde;
##STR15## where X is a fluorophore or hydrogen, and in which the 3'"'"' hydroxyl group can be liberated in two steps which are;oxidation of the vicinal diol by periodate; and elimination as a result of the action of hydrazine. - View Dependent Claims (18, 19)
-
Specification