Non-radioactive labelled single-stranded probe, method for manufacturing it and method for detecting a specified nucleotide sequence using this probe

• US 4,978,749 A
• Filed: 07/11/1989
• Issued: 12/18/1990
• Est. Priority Date: 04/04/1986
• Status: Expired due to Fees

• Current Assignee: Centre National De La Recherche Scientifique, Institut Pasteur
• Original Assignee: Centre National De La Recherche Scientifique, Institut Pasteur
• Inventors: Traincard, Francois, Vo Quang, Tuyen, Stratis, Avrameas, Sakamoto, Hiroshi, Ternynck, Therese, Guesdon, Jean-Luc
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Marschel, Ardia
• Application Number:
  US07/378,507
• Time in Patent Office: 525 Days
• Field of Search: 435/6, 435/235, 435/239, 435/320, 435/7, 436/501, 530/387, 536/27, 935/73, 935/12, 935/78, 935/110
• US Class Current: 536/24.3
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6813 Hybridisation assays

Abstract

The invention relates to a new probe for detecting a specified nucleic acid sequence. It consists of a recombinant DNA containing an insert complementary to the nucleic acid sequence to be detected and nuclecic acid sequences derived from the DNA (+) of phage M13, the bases which participate in the composition of this recombinant DNA, in particular the thymine groups, being replaced by 5-bromouracil groups. The invention also relates to a method for manufacturing this probe, this method comprising the culturing of cell hosts, more especially E. coli, which are auxotrophic for thymine in a medium containing a limiting concentration of thymine and containing 5-bromouracil. After the requisite incubation time, the supernatant is removed and the probe is recovered from the bacterial cells.

Claims

1. A recombinant probe comprising a single-stranded nucleic acid having a single polarity and

(a) a first nucleotide sequence for hybridization with another nucleotide sequence that is to be detected, and

(b) a second nucleotide sequence that comprises a nucleotide sequence of a single-stranded phage,

wherein said recombinant probe comprises a plurality of a modified base, said modified base comprising a corresponding natural base modified by a halogenated substituent, and

wherein the number of said modified bases in said nucleic acid constitutes more than 60% of the sum of the modified bases and said corresponding natural base, and

wherein said nucleic acid is capable of replication in a host cell, and capable of existing in a double-stranded form during replication in the host cell, and said double-stranded form is incapable of being excreted outside the host cell.

2. A recombinant probe as claimed in claim 1, wherein all of said corresponding natural base in the nucleic acid is modified by the halogenated substituent.

3. A recombinant probe as claimed in claim 1, wherein said phage is M13.

4. A recombinant probe as claimed in claim 1, wherein said phage is φ

x174.

5. A recombinant probe as claimed in claim 1, wherein said corresponding natural base is thymine and said modified base is 5-bromouracil.

6. A recombinant probe as claimed in claim 1, wherein said host cell is E. coli.

7. A recombinant probe as claimed in claim 1, wherein said probe is capable of reacting immunologically with monoclonal antibodies directed to said modified base.

8. A recombinant probe as claimed in claim 1, wherein said first nucleotide sequence is an insert in a region of the second nucleotide sequence, and wherein said region is not essential for replication of the recombinant probe in the host cell.

9. A recombinant probe as claimed in claim 1, wherein said host cell is auxotrophic for said corresponding natural base.