Process for the production of modified nucleic acids

• US 5,741,637 A
• Filed: 06/06/1994
• Issued: 04/21/1998
• Est. Priority Date: 01/17/1990
• Status: Expired due to Fees

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Kessler, Christoph, Rueger, Rudiger
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/254,422
• Time in Patent Office: 1,415 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.18
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Process for the production of nucleic acids by hybridization of at least one primer to a template nucleic acid and enzymatic elongation of the primer to a nucleic acid strand which is complementary to the template nucleic acid by reaction with different types of nucleoside triphosphates, wherein the complementary nucleic acid strand carries two or more groups which enable it to be immobilized and one or more detectable groups, a method which uses this process for the detection of nucleic acids, suitable reagent kits therefor and modified nucleic acids.

Claims

1. A process for the production of labelled nucleic acids comprising the steps of

a) hybridizing at least one primer to a template nucleic acid, and

b) enzymatically elongating the primer by reaction with a set of nucleoside triphosphates to produce a newly formed complementary nucleic acid strand having at least two immobilizable biospecific labels and at least one detectable biospecific label,

wherein said set of nucleoside triphosphates comprises at least one type of nucleoside triphosphate with a detectable, biospecific label, the same or at least one other type of nucleoside triphosphate with an immobilizable, biospecific label and unmodified nucleoside triphosphates, and wherein said immobilizable biospecific label is different from said detectable biospecific label.

2. The process according to claim 1, wherein said primer contains at least one immobilizable, biospecific label.

3. The process according to claim 1, wherein newly formed complementary nucleic acids formed in step b) are incorporated into step a) to serve as a template nucleic acid.

4. The process according to claim 1, wherein said set of nucleoside triphosphates comprises detectably labelled nucleoside triphosphates, immobilizably labeled nucleoside triphosphates and unmodified nucleoside triphosphates in a mixture ranging from 1:

1;

1 to 1;

1;

2, wherein said unmodified nucleoside triphosphates include all 4 types of natural nucleoside triphosphates.

6. The process according to claim 1, wherein said primer contains at least one detectable biospecific label.

7. The process according to claim 1, wherein the immobilizable, biospecific label of at least one type of nucleoside triphosphates is biotin.

8. The process according to claim 1, wherein the detectable biospecific label of at least one type of nucleoside triphosphates is digoxigenin.

9. The process according to claim 1, wherein said primer contains at least one detectable, biospecific label, and said set of nucleoside triphosphates contains at least one type of nucleoside triphosphate with biotin as an immobilizable, biospecific label and at least one type of nucleoside triphosphate with digoxigenin as a detectable biospecific label.

10. The process according to claim 1, wherein said primer contains at least one immobilizable label, and said set of nucleoside triphosphates contains at least one type of nucleoside triphosphate with biotin as an immobilizable, biospecific label, and at least one type of nucleoside triphosphate with digoxigenin as a detectable biospecific label.

11. The process according to claim 1, wherein said set of mononucleotides contains at least one type of mononucleotide in the unmodified form and the same type of mononucleotide with a detectable biospecific label or with an immobilizable, biospecific label.

12. The process according to claim 1, wherein said primer is unmodified at its 3'"'"'-end.

5. A process for the production of labelled nucleic acids comprising the steps of

a) separating a nucleic acid into single strands which are used as template nucleic acids,

b) hybridizing at least one primer to said template nucleic acids,

c) enzymatically elongating said at least one hybridized primer in the presence of a set of different types of nucleoside triphosphates producing an elongation product having at least two immobilizable labels of the same type and at least one detectable label, wherein said elongation product is complementary to the template nucleic acid, and

d) amplifying said elongation product by repeating steps a), b) and c) in the presence of said set of different types of modified nucleoside triphosphates wherein at least one type of said nucleoside triphosphates is bound to a detectable biospecific label and the same or at least one other type of nucleoside triphosphate is bound to an immobilizable biospecific label,

wherein said immobilizable biospecific label is different from said detectable biospecific label.

13. A process for the detection of a nucleic acid comprising the steps of:

a) hybridizing at least one primer to the nucleic acid,

b) enzymatically elongating the primer by reaction with a set of nucleoside triphosphates to produce a newly formed complementary nucleic acid strand having at least two immobilizable biospecific labels and at least one detectable biospecific label wherein said set of nucleoside triphosphates comprises at least one type of nucleoside triphosphate with a detectable, biospecific label, the same or at least one other type of nucleoside triphosphate with an immobilizable biospecific label and unmodified nucleoside triphosphates,

c) immobilizing the newly formed complementary nucleic acid by means of the at least two immobilizable biospecific labels, and

d) detecting immobilization by means of the at least one detectable biospecific label.