Patent EP0212546A2 successfully refreshed from Google Patents.
EP0212546A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Method for labeling polynucleotide sequences
Assignee: Enzo Biochem Inc
•
Filing: 1986-08-12
•
Granted: —
•
Expires: 2006-08-12
Abstract
Abstract A method for labeling a polynucleotide sequence to form a polynucleotide probe is disclosed. The method comprises forming a modified polynucleotide sequence wherein the modification comprises the cleavage of preselected bases to generate sugar aldehydes. The preselected bases are either qua- temized nitrogen purines or uracil. A signalling moiety is attached to the modified polynucleotide sequence by means of the sugar aldehydes.
Inventors
- 👤 Jannis Stavrianopoulos
- 👤 Elazar Rabbani
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-01 18:34
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
59 words
A method of labeling an alkylated polynucleotide sequence comprising generating a sugar aldehyde by cleaving a quaternized nitrogen purine nucleotide from said sequence at a pH from about 5.0 to about 7.5 and at a temperature from about 35°C to about 60°C in the presence of a signalling moiety whereby said signalling moiety becomes attached to said sugar aldehyde.
Claim 2
Independent Claim
59 words
A method of labeling an alkylated polynucleotide sequence comprising generating a sugar aldehyde by cleaving a quaternized nitrogen purine nucleotide from said sequence at a pH from about 5.0 to about 7.5 and at a temperature from about 35°C to about 60°C in the presence of a signalling moiety whereby said signalling moiety becomes attached to said sugar aldehyde.
Claim 3
Dependent Claim
38 words
The method of Claim 1 wherein said quaternized nitrogen purine nucleotide in said modified sequence is obtained by contacting a polynucleotide sequence with an alkylating agent or by incorporating a quaternized nitrogen purine nucleotide into a polynucleotide sequence.
Claim 4
Dependent Claim
22 words
The method of Claim 2 wherein said quaternized nitrogen purine nucleotide is formed by contacting a purine nucleotide with an alkylating agent.
Claim 5
Dependent Claim
33 words
The method of Claim 2 or 3 wherein said alkylating agent is selected from the group consisting of diazomethane, dialkyl sulfates, dialkyl sulfonates, and alkyl halides, preferably, diazomethane, dimethyl sulfate, and methyl iodide.
Claim 6
Dependent Claim
31 words
The method of Claim 3 or 4 wherein said quaternized nitrogen purine nucleotide is 7-methylguanylic acid or 7-methyladenylic acid and is incorporated into said polynucleotide sequence by means of nick translation.
Claim 7
Independent Claim
63 words
A method of labeling a polynucleotide sequence comprising the steps of: (a) cleaving at least one uracil base from said polynucleotide sequence with hydroxylamine to generate a sugar oxime at the 1-position of said sugar; (b) contacting said polynucleotide sequence with a ketone-containing compound to covert said sugar oxime to a sugar aldehyde; and (c) attaching a signalling moiety to said sugar aldehyde.
Claim 8
Independent Claim
63 words
A method of labeling a polynucleotide sequence comprising the steps of: (a) cleaving at least one uracil base from said polynucleotide sequence with hydroxylamine to generate a sugar oxime at the 1-position of said sugar; (b) contacting said polynucleotide sequence with a ketone-containing compound to covert said sugar oxime to a sugar aldehyde; and (c) attaching a signalling moiety to said sugar aldehyde.
Claim 9
Dependent Claim
20 words
The method of Claim 6 wherein said polynucleotide sequence is DNA and said uracil base is incorporated as deoxyuridylic acid.
Claim 10
Dependent Claim
56 words
The method of Claim 6 or 7 wherein said cleaving step is-carried out in a 10M hydroxylamine solution, wherein the temperature of said solution is from about 0°C to about 40°C, preferably from about 5°C to about 15°C, and more preferably about 10°C and the pH of said solution is from about 9.0 to about 11.0.
Claim 11
Independent Claim
34 words
The method of any one of claims 6 to 8 wherein said contacting step is carried out in a solution wherein the concentration of said ketone-containing compound is from about 0.1M to about 0.5M.
Claim 12
Independent Claim
31 words
The method of any one of the preceding claims wherein said signalling moiety comprises a functional group selected from the group consisting of NH 2 -NH-, NH 2 -NH-C-, and HS.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.