Use of extremely thermophilic DNA-polymerases

• US 6,251,637 B1
• Filed: 04/07/1999
• Issued: 06/26/2001
• Est. Priority Date: 03/29/1996
• Status: Expired due to Fees

• Current Assignee: Gsf-Forschungszentrum Fur Umwelt und Gesundheit
• Original Assignee: Gsf-Forschungszentrum Fur Umwelt und Gesundheit
• Inventors: Blusch, Juergen
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/155,512
• Time in Patent Office: 811 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 536/23.1, 935/77
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

Cycle sequencing of DNA with marker-labeled dideoxynucleotides is performed using extremely thermophilic DNA polymerases in a one-lane technique in which dideoxynucleotides labeled with a dye as a marker are used to obtain sequence ladders of more 500 bases. Chain elongation is performed at a temperature of 65 to 75° C. Surprisingly, it was found that chain elongation temperatures of substantially more than 60° C. could be used in cycle sequencing of DNA with marker labeled dye dideoxynucleotides when using extremely thermophilic DNA polymerases.

Claims

1. In a method for cycle sequencing of DNA in the form a 1-lane technique using dideoxynucleotides labeled with a dye as a marker, wherein sequence ladders of more than 500 bases are obtained, comprising the steps of:

a) mixing at least a reaction buffer, deoxynucleotides, the marker-labeled dideoxynucleotides, primers and double stranded DNA template, water and optionally further additives and adjusting the concentration thereof in relationship to each other;

b) adding at least one extremely thermostable DNA polymerase,

c) performing PCR sequencing cycles comprising the steps of;

denaturing the template DNA at an appropriate temperature;

annealing the primer at an appropriate temperature;

chain elongation at an appropriate temperature; and

adjusting the ramping time between the individual temperature stages in dependence on the DNA polymerase, template/primer pair and reaction buffer used;

d) purifying the reaction sample; and

e) separating the DNA chains and detecting to determine the DNA sequence;

the improvement comprising;

(i) in step c) performing the chain elongation at a temperature of 65°

C. to 75°

C.;

(ii) depending on the primer/template pair and DNA polymerase used, adjusting the following conditions in relationship to each other to enable chain elongation in a temperature range of 65°

C. to 75°

C.;

concentration of metal ions;

concentration of nucleotides;

concentration of DNA polymerase; and

the conditions for primer annealing as well as conditions for transition from primer annealing to chain elongation temperature; and

(iii) adding additives for stimulation of primer annealing, chain elongation and DNA polymerase activity.

2. Method according to claim 1, wherein the buffer contains alkaline metal ions or alkaline earth metal ions.

3. Method according to claim 2, wherein magnesium ions are used as said alkaline earth metal ions.

4. Method according to claim 1, wherein in step c) the chain elongation is performed at a temperature in the range of 68°

C. to 73°

C.

5. Method according to claim 1, wherein in step c) the chain elongation is performed at a temperature in the range of 70°

C. to 72°

C.

6. Method according to claim 1, further comprising adding polyethylene glycol, stabilizers of DNA structure, or both, in step a).

7. Method according to claim 1, wherein dideoxynucleotides labeled with a fluorescent dye are used as a marker.

8. Method according to claim 1, wherein spermidine is added as a stabilizer of DNA structure.