DNA sequencing method

• US 6,087,095 A
• Filed: 12/09/1994
• Issued: 07/11/2000
• Est. Priority Date: 04/22/1992
• Status: Expired due to Term

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Rosenthal, Andre, Brenner, Sydney
• Primary Examiner(s): Degen, Nancy
• Assistant Examiner(s): McGarry, Sean
• Application Number:
  US08/325,224
• Time in Patent Office: 2,041 Days
• Field of Search: 435/6, 435/41, 435/172.1, 935/76, 935/77, 935/78, 536/23.1, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/149 Sequential reactions

Abstract

The invention is drawn to a method of DNA sequencing using labeled nucleotides that do not act as chain elongation inhibitors where the label is removed or neutralized for the sequential addition of non-labeled nucleotides.

Claims

1. A method for determining the sequence of a nucleic acid comprising the steps of:

(a) forming a single-stranded template comprising the nucleic acid to be sequenced;

(b) hybridizing a primer to the template to form a template/primer complex;

(c) extending the primer by the addition of a single labelled nucleotide which is not a chain elongation inhibitor;

(d) determining the type of the labelled nucleotide added onto the primer;

(e) removing or neutralizing the label; and

(f) repeating steps (c) to (e) sequentially and recording the order of incorporation of labelled nucleotides.

2. The method according to claim 1 wherein the template/primer complex of said step (b) is bound to a solid-phase support.

3. The method according to claim 1 wherein said extending step (c) further comprises the addition of unlabelled nucleotides.

4. The method according to claim 1 wherein said extending step (c) is carried out in the presence of a non-labelled chain elongation inhibitor.

5. The method according to claim 4, wherein said non-labelled chain elongation inhibitor is incorporated into the template/primer complex, and step (e) further comprises removing said non-labelled chain elongation inhibitor.

6. The method according to claim 4 wherein said non-labelled chain elongation inhibitor is not incorporated into said template/primer complex.

7. The method according to claim 6 wherein said non-labelled chain elongation inhibitor is selected from the group consisting of deoxynucleoside 5'"'"'-[α

,β

-methylene]triphosphates, deoxynucleoside diphosphates, and deoxynucleoside monophosphates.

8. The method according to claim 1 wherein said template/primer complex comprises a primer linked through its 3'"'"' terminus to a deoxynucleoside phosphorothioate base.

9. The method according to claim 8 wherein step (e) comprises:

i) removing the labelled nucleotide with an exonuclease; and

ii) replacing the labelled nucleotide with a corresponding unlabelled phosphorothioate nucleoside derivative in the presence of a non-labelled chain elongation inhibitor.

10. The method according to claim 1 wherein said steps (c) and (d) are repeated sequentially a multiplicity of times before said step (e).

11. The method according to claim 1 wherein the label is a fluorescent label and said step (e) comprises a process selected from the group consisting of neutralizing the label by bleaching with laser radiation, neutralizing the label using a chemical, and dissociating the label from the labelled nucleotide.

12. A process for sequencing a DNA fragment comprising the steps of:

i) hybridizing a capped primer comprising a phosphorothioate nucleoside derivative to a template to form a template/primer complex;

ii) adding a labelled deoxynucleoside triphosphate together with a mixture of non-labelled heterogenous chain terminators and a polymerase to the template/primer complex, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor;

iii) removing excess reagents by washing;

iv) treating the template/primer complex with an exonuclease to remove both the labelled nucleotide resulting from incorporation of said labelled deoxynucleoside, and the non-labelled chain terminators;

vi) removing the exonuclease by washing;

vii) adding a phosphorothioate deoxynucleoside triphosphate corresponding to the labelled deoxynucleoside triphosphate added in step (ii) together with a mixture of non-labelled heterogeneous chain terminators;

viii) removing excess reagents by washing;

ix) treating the template/primer complex with an exonuclease to remove the non-labelled chain terminators; and

x) removing the exonuclease by washing.

13. A process for sequencing a DNA fragment comprising the steps of:

i) hybridizing a capped primer containing a phosphorothioate nucleoside derivative to a template to form a template/primer complex;

ii) adding a labelled deoxynucleotide together with non-labelled heterogeneous chain terminators and a suitable polymerase, wherein said labelled deoxynucleotide is not a chain elongation inhibitor;

iii) removing excess reagents by washing;

iv) measuring the a mount of incorporated label;

v) removing the labelled nucleotide and the terminators with an exonuclease;

vi) removing the exonuclease by washing;

vii) adding a phosphorothioate deoxynucleotide together with a mixture of non-labelled heterogeneous chain elongation inhibitors not incorporated into the chain; and

viii) removing excess reagents by washing.

14. A process for sequencing a DNA fragment comprising the steps of:

i) hybridizing a capped primer containing a phosphorothioate deoxynucleotide to a template to form a template/primer complex;

ii) adding a labelled nucleotide triphosphate together with a mixture of non-labelled heterogenous chain elongation inhibitors not incorporated into the chain, wherein said labelled nucleotide triphosphate is not a chain elongation inhibitor;

iii) removing excess reagents by washing;

iv) measuring the amount of incorporated label;

v) repeating said steps ii, iii and iv sequentially until all four different labelled nucleotides in the presence of their corresponding, non-labelled heterogeneous chain elongation inhibitors not incorporated into the chain have been added;

vi) removing all labelled nucleotides with exonuclease;

vii) removing the exonuclease by washing;

viii) adding the phosphorothioate deoxynucleotide corresponding to the first labelled deoxynucleotide added to the reaction in said step ii, together with non-labelled heterogenous chain elongation inhibitors not incorporated into the chain and a polymerase;

ix) removing excess reagents by washing; and

x) repeating said steps viii and ix with the three remaining phosphorothioate deoxynucleoside derivatives.

15. A process for sequencing a DNA fragment comprising the steps of:

i) hybridizing a capped primer to a template to form a template/primer complex;

ii) adding a fluorescent nucleoside triphosphate, together with three non-labelled heterogeneous chain elongation inhibitors not incorporated into the chain and a polymerase, wherein said fluorescent nucleoside triphosphate is not a chain elongation inhibitor;

iii) removing excess reagents by washing;

iv) measuring the amount of incorporated label;

v) repeating said steps ii, iii, and iv using all three different nucleoside triphosphates, each with a fluorescent label, in the presence of the respective non-labelled heterogeneous chain elongation inhibitors not incorporated into the chain; and

vi) destroying the fluorescent labels by bleaching with a laser of by a suitable chemical reaction, or removing the fluorescent labels by a chemical cleavage step.

16. A DNA sequencing kit comprising:

i) a linker for attaching a DNA template to a solid-phase matrix the liner comprising a primer having a phosphorothioate deoxynucleoside residue at its 3'"'"' end;

ii) non-labelled chain elongation inhibitors;

iii) labelled nucleoside triphosphates which are not chain elongation inhibitors;

iv) phosphorothioate deoxynucleoside triphosphates;

v) a 5'"'"'→

3'"'"' DNA polymerase; and

vi) a 3'"'"'→

5'"'"' exonuclease.