Method

• US 20050084851A1
• Filed: 09/10/2001
• Published: 04/21/2005
• Est. Priority Date: 09/08/2000
• Status: Abandoned Application

• Current Assignee: Biotage AB (Biotage)
• Original Assignee: Biotage AB (Biotage)
• Inventors: Pourmand, Nader, Ekstorm, Bjorn, Ronaghi, Mostafa
• Application Number:
  US10/363,177
• Publication Number:
  US 20050084851A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2537/149 Sequential reactions

Abstract

The method of the invention relates to a method of typing one or more nucleic acid molecules, said method comprising: simultaneously or sequentially performing two or more primer extension reactions, each primer binding at a different predetermined site in said nucleic acid molecule(s), and determining the pattern of nucleotide incorporation to obtain a test pattern for said nucleic acid molecule(s) which is optionally compared with one or more reference patterns to type the said nucleic acid molecule(s).

Claims

1. A method of typing one or more nucleic acid molecules, said method comprising:

simultaneously hybridizing two or more extension primers to said nucleic acid molecule or molecules and performing primer extension reactions therefrom, each primer binding at a different predetermined site in said nucleic acid molecule or molecules, and determining the pattern of nucleotide incorporation by sequencing-by-synthesis to obtain a test pattern for said nucleic acid molecule or molecules which is optionally compared with one or more reference patterns to type the said nucleic acid molecule or molecules.

2. A method as claimed in claim 1 wherein the nucleic acid contains two or more variable sites.

5. A method as claimed in claim 1 wherein nucleotides are added to the reaction mix sequentially in a predetermined order.

6. A method as claimed in claim 1 wherein nucleotide incorporation is determined quantitatively.

7. A method as claimed in claim 1 wherein if nucleotide incorporation takes place at one variable site, there is no nucleotide incorporation at the other variable site(s).

8. A method as claimed in claim 1 wherein a first extension primer binds closer to its variable site than a second primer does to its variable site.

9. A method as claimed in claim 8 wherein the second primer is 10-20 nucleotides further away from its variable site than is said first primer.

10. A method as claimed in claim 1 wherein single-stranded binding protein is added to the reaction mix after the primers are annealed to the nucleic acid template.

11. A method as claimed in claim 1 wherein the primer extension reactions occur simultaneously.

12. A method as claimed in claim 1 wherein 3 or more variable sites are typed.

13. A method as claimed in claim 1 wherein 3 or more primer extension reactions are performed.

3. A method for obtaining typing information about a plurality of variable sites within target nucleic acid, comprising simultaneously hybridizing two or more extension primers to said nucleic acid and performing primer extension reactions therefrom, each primer binding at a different predetermined site in said target nucleic acid, the pattern of nucleotide incorporation determined from said primer extension reactions by sequencing-by-synthesis providing the typing information about said variable sites.

4. (Cancelled).

14. A method of diagnosis of pathological conditions characterised by the presence of specific nucleic acid molecule or molecules, comprising simultaneously hybridizing two or more extension primers to said nucleic acid molecule or molecules, and performing primer extension reactions therefrom, each primer binding at a different predetermined site in said nucleic acid molecule or molecules, the pattern of nucleotide incorporation, determined from said primer extension reactions by sequencing-by-synthesis, allowing diagnosis of said pathological conditions.

15. A kit for use in a method of typing nucleic acid which comprises:

optionally one or more primers for in vitro amplification;

two or more primers for primer extension reactions each primer binding at a different predetermined site in a nucleic acid molecule;

nucleotides for amplification and/or for the primer extension reaction;

optionally a polymerase enzyme for the amplification and/or primer extension reaction; and

optionally means for detecting primer extension.