Method for making nucleic acid probes
First Claim
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1. A method of making a polynucleotide probe, comprising:
- (a) providing a modified nucleotide given by the structure ##STR21## wherein;
R1 is a protected or unprotected --NH2, --COOH or --SH group;
R2 is a bond or an amide, thioether or disulfide linking moeity;
R3 is hydrogen, methyl, bromine, fluorine or iodine;
R4 is selected from the group consisting of hydrogen, acid-sensitive, base-stable blocking groups and acyl capping groups;
R5 is a phosphorus derivative selected from the group consisting of PO3 H2, phosphotriesters, phosphodiesters, phosphites, phosphoramidites, H-phosphonates and phosphorothioates and may be bound through R5 to a solid support;
R6 is H, OH, or OR where R is an acid-sensitive, base-stable protecting group; and
x is an integer in the range of 1 and 8 inclusive; and
(b) sequentially adding nucleotides to either the 3'"'"' and of said modified nucleotde, the 5'"'"' end, or both, wherein a fraction of the added nucleotides comprise a plurality of said modified nucleotides, thereby providing a polynucleotide probe having said modified nucleotides incorporated at predetermined, spaced apart positions.
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Abstract
Modified nucleotides are provided which have the structure ##STR1## wherein R1 is a reactive group derivatizabale with a detectable label, R2 is an optional linking moiety including an amide, thioether or disulfide linkage or a combination thereof. R3 is hydrogen, methyl, bromine, fluorine or iodine, R4 is hydrogen, an acid-sensitive, base-stable blocking group or an acyl capping group, R5 is hydrogen or a phosphorus derivative, R6 is H, OH, or OR where R is a protecting group and x is an integer in the range of 1 and 8 inclusive. Methods of synthesizing the derivatizable nucleotide are disclosed, as are labeled polynucleotide probes prepared therefrom.
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Citations
6 Claims
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1. A method of making a polynucleotide probe, comprising:
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(a) providing a modified nucleotide given by the structure ##STR21## wherein;
R1 is a protected or unprotected --NH2, --COOH or --SH group;R2 is a bond or an amide, thioether or disulfide linking moeity; R3 is hydrogen, methyl, bromine, fluorine or iodine; R4 is selected from the group consisting of hydrogen, acid-sensitive, base-stable blocking groups and acyl capping groups; R5 is a phosphorus derivative selected from the group consisting of PO3 H2, phosphotriesters, phosphodiesters, phosphites, phosphoramidites, H-phosphonates and phosphorothioates and may be bound through R5 to a solid support; R6 is H, OH, or OR where R is an acid-sensitive, base-stable protecting group; and x is an integer in the range of 1 and 8 inclusive; and (b) sequentially adding nucleotides to either the 3'"'"' and of said modified nucleotde, the 5'"'"' end, or both, wherein a fraction of the added nucleotides comprise a plurality of said modified nucleotides, thereby providing a polynucleotide probe having said modified nucleotides incorporated at predetermined, spaced apart positions. - View Dependent Claims (2, 3, 4, 5, 6)
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Specification