Synthesis of fluorophore-labeled DNA

• US 5,945,312 A
• Filed: 11/07/1997
• Issued: 08/31/1999
• Est. Priority Date: 04/15/1996
• Status: Expired due to Fees

• Current Assignee: University of Alberta, University of Southern California
• Original Assignee: University of Alberta, University of Southern California
• Inventors: Goodman, Myron F., Reha-Krantz, Linda J.
• Primary Examiner(s): Knode, Marian C.
• Assistant Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US08/966,145
• Time in Patent Office: 662 Days
• Field of Search: 435/6, 435/91.1, 435/220, 436/94, 436/173, 536/23.1, 536/24.3
• US Class Current: 435/91.1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

Sequencing methods and methods for synthesizing DNA probes using mutant bacteriophage T4 DNA polymerases which have increased ability to incorporate modified nucleotides for the synthesis of long or short chains of complementary, modified, e.g., fluorophore-labeled DNA. In general, the mutant T4 DNA polymerases retain 3'"'"'→5'"'"' exonuclease activity; hence, reduction or elimination of 3'"'"'→5'"'"' exonuclease activity is not a prerequisite for efficient synthesis of a complementary fluorophore-labeled or other modified DNA. In fact, retention of 3'"'"'→5'"'"' exonuclease activity increases accuracy of DNA replication, because these exonucleases proofread or edit the product of DNA replication.

Claims

1. A method for synthesizing a complementary DNA, from a DNA template, the improvement comprising:

using a mutant Family B DNA polymerase, with increased intrinsic processivity relative to native DNA polymerase, to synthesize a complementary DNA having one or more fluorophore-labeled nucleotides incorporated therein.

2. The method of claim 1, wherein the mutant Family B polymerase is a mutant T4 DNA polymerase selected from the group consisting of L412M-DNA polymerase, Q380K-DNA polymerase, E395K-DNA polymerase, E743K-DNA polymerase, M725I-DNA polymerase, M725V-DNA polymerase, S756P-DNA polymerase, L771F-DNA polymerase, L771H-DNA polymerase, -DNA polymerase, -DNA polymerase, V355A-DNA polymerase, E395K+L412M-DNA polymerase, L412M+E473K-DNA polymerase, E395K+L412M+E743K-DNA polymerase, and Q380K+L412M+E743K-DNA polymerase.

3. The method of claim 1, wherein the mutant Family B polymerase is a multiple mutant T4 DNA polymerase with one or more amino acid substitutions selected from the group consisting of:

Q380K, E395K, E743K, M725I, M725V, S756P, L771F, L771H, and V355A.

4. The method of claim 1, further comprising the step of employing the resulting complementary DNA in single molecule sequencing, making DNA probes, or mass spectrometry sequencing.

5. The method of claim 1, wherein the mutant DNA polymerase incorporates at least two different fluorophore-labeled nucleotides into complementary DNA.

6. The method of claim 1 wherein one or more unmodified nucleotides are replaced by the corresponding fluorophore-labeled nucleotides.

7. The method of claim 1, wherein the one or more fluorophore-labeled nucleotides are selected from the group consisting of fluorescein- and rhodamine-labeled nucleotides.

8. The method of claim 1 wherein the mutant Family B DNA polymerase is selected from the group consisting of T2, T4, and T6 mutant DNA polymerases.

9. The method of claim 1 wherein the mutant Family B DNA polymerase has an amino acid substitution in highly conserved Motif A.

10. The method of claim 1, wherein the polymerase is a mixture comprising a first polymerase and a second polymerase, wherein:

a. the first polymerase is a mutant T2, T4, or T6 DNA polymerase selected from the group consisting of L412M-DNA polymerase, Q380K-DNA polymerase, E395K-DNA polymerase, E743K-DNA polymerase, M725I-DNA polymerase, M725V-DNA polymerase, S756P-DNA polymerase, L771F DNA polymerase, L771H-DNA polymerase, -DNA polymerase, -DNA polymerase, V355A-DNA polymerase, E395K+L412M-DNA polymerase, L412M+E473K-DNA polymerase, E395K+L412M+E743K-DNA polymerase, and Q380K+L412M+E743K-DNA polymerase; and

b. the second polymerase is selected from the group consisting of an exonuclease deficient DNA polymerase, and a thermostable DNA polymerase.

11. The method of claim 1, wherein the DNA template is primed with a first oligonucleotide.

12. The method of claim 11, wherein further synthesis of the complementary DNA is blocked by a second oligonucleotide.

13. The method of claim 1, wherein the complementary DNA is a full-length copy of the template DNA.

14. The method of claim 1 wherein the DNA template is covalently joined to a self-annealing hairpin DNA.

15. The method of claim 14, wherein the hairpin DNA contains at least one nonhydrolyzable internucleotide linkage.

16. The method of claim 15, wherein the internucleotide linkage is a phosphorothioate linkage.

17. The method of claim 14, wherein the hairpin DNA further comprises means for immobilizing a DNA molecule in a flow cell.

18. The method of claim 17, wherein the hairpin DNA contains one or more biotin residues.

19. The method of claim 1, wherein synthesizing the complementary DNA gives a duplex DNA in which one strand of the duplex DNA contains fluorophore-labeled DNA.

20. The method of claim 19, wherein the DNA template is covalently joined to a self-annealing hairpin DNA.

21. The method of claim 20, wherein the hairpin DNA further comprises means to anchor duplex DNA to a streptavidin-coated bead.

22. The method of claim 1, further comprising immobilizing the complementary DNA in a flow cell and subjecting the complementary DNA to exonuclease digestion.

23. The method of claim 22, further comprising detecting the fluorophore-labeled nucleotides released by the exonuclease digestion.