Method for making nucleic acid probes

• US 4,910,300 A
• Filed: 05/05/1988
• Issued: 03/20/1990
• Est. Priority Date: 12/11/1985
• Status: Expired due to Term

• Current Assignee: Chiron Diagnostics Corp (Bayer AG)
• Original Assignee: Chiron Corporation (Novartis Ag)
• Inventors: Urdea, Michael S., Horn, Thomas
• Primary Examiner(s): Rosen, Sam
• Application Number:
  US07/190,698
• Time in Patent Office: 684 Days
• Field of Search: 536/24, 536/27, 536/28, 435/6
• US Class Current: 536/26.8
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/706 for hepatitis
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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.

Claims

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.

2. The method of claim 1, further including, after (b):

(c) labeling the modified nucleotides within said polynucleotide probe by derivatization with a labeling species at the R¹ moiety.

3. The method of claim 1, further including, prior to step (a), labeling each of said modified nucleotides to be incorporated into said probe by derivatization with a labeling species at the R¹ moiety.

4. The method of claim 1, wherein R¹ is a protected or unprotected --NH₂ group.

5. The method of claim 4, wherein R² is a bond or an amide linking moiety.

6. The method of claim 5, wherein R⁴ and R⁶ are hydrogen.