Sequencing method using magnifying tags

• US 6,723,513 B2
• Filed: 06/22/2001
• Issued: 04/20/2004
• Est. Priority Date: 12/23/1998
• Status: Expired due to Fees

• Current Assignee: Empire IP LLC
• Original Assignee: Lingvitae AS
• Inventors: Lexow, Preben
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/886,223
• Publication Number:
  US 20020028458A1
• Time in Patent Office: 1,033 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 506/4
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

The present invention provides a method of sequencing all or part of a target nucleic acid molecule by determining the sequence of a portion of said nucleic acid molecule in addition to information regarding the position of said portion and in particular provides a new method of sequencing involving the magnification of one or more bases of said bases to aid identification.

Claims

1. A method of magnifying a signal associated with one or more nucleotide bases of a nucleotide sequence in a target nucleic acid molecule comprising the steps of:

(A) treating said target nucleic acid molecule so that at least a region of said target nucleic acid molecule is converted into an adapter binding region which is a region suitable for binding an adapter molecule;

wherein said adapter molecule comprises;

(i) one or more magnifying tags, or

(ii) a means for attaching one or more of said magnifying tags to said adapter binding region,

wherein said magnifying tag(s) correspond to;

(a) one or more nucleotide bases of the adapter binding region or (b) one or more nucleotide bases adjacent to the adapter binding region or (c) one or more nucleotide bases which overlap the adapter binding region;

(B) binding said adapter molecule to at least a portion of said adapter binding region created in step (A) to form a nucleic acid molecule;

adapter molecule complex;

(C) optionally, ligating said target nucleic acid molecule to said adapter molecule such that at least said magnifying tag(s) remain associated with said target nucleic acid molecule and to form a ligated nucleic acid molecule;

adapter molecule complex;

(D) treating the resulting complex of step (B) or the resulting complex of step (C) so that at least another region of said target nucleic acid molecule is converted into a form suitable for binding another adapter molecule, wherein said another region comprises one or more nucleotide bases which are not associated with the magnifying tags of step (B); and

thereafter

(E) repeating steps (B) to (D), with the proviso that the adapter molecule in each cycle of steps (B) to (C) binds to a region adjacent to a region of said target nucleic acid molecule to which the adapter molecule of a previous cycle bound, or the adapter molecule in each cycle of steps (B) to (C) binds to a region which overlaps with a region of said target nucleic acid molecule to which the adapter molecule of a previous cycle bound, and wherein the magnifying tags of each cycle of steps (A) to (C) are ligated together, to thereby magnify said signal.

2. The method as claimed in claim 1, wherein in step (A), said form is a single-stranded nucleic acid molecule.

3. The method as claimed in claim 1, wherein said magnifying tag(s) correspond to one or more nucleotide bases of said adapter binding region.

4. The method as claimed in claim 1, wherein each magnifying tag corresponds to at least 2 nucleotide bases in said adapter binding region or to at least 2 nucleotide bases adjacent to said adapter binding region.

5. The method as claimed in claim 1, wherein said magnifying tags together correspond to at least 2 nucleotide bases in said adapter binding region or to at least 2 nucleotide bases adjacent to said adapter binding region.

6. The method as claimed in claim 5, wherein said magnifying tags together correspond to at least 4 nucleotide bases in said adapter binding region or to at least 4 nucleotide bases adjacent to said adapter binding region.

7. The method as claimed in claim 1, wherein a chain of magnifying tags are associated with said target nucleic acid molecule.

8. The method as claimed in claim 7, wherein said chain comprises 4 or more magnifying tags corresponding to at least 4 contiguous nucleotide bases.

9. The method as claimed in claim 1, wherein said magnifying tags are nucleic acid sequences of at least 2 nucleotide bases in length.

10. The method as claimed in claim 9, wherein said magnifying tags are nucleic acid sequences of 10 to 30 nucleotide bases in length.

11. The method as claimed in claim 1, wherein said adapter molecule comprises a recognition site for a nuclease, which has a cleavage site separate from its recognition site.

12. The method as claimed in claim 1, wherein said adapter molecule comprises recognition sites for 2 or more nucleases, which have cleavage sites separate from their respective recognition sites, wherein cleavage with said nucleases produces single-stranded regions which are adjacent or overlapping.

13. The method as claimed in claim 1, wherein two or more adapter molecules are bound in step (B).

14. The method as claimed in claim 13, wherein said adapter molecules are bound to overlapping or adjacent regions.

15. The method as claimed in claim 14, wherein said adapter molecules are bound to overlapping regions thereby allowing the association of more than one magnifying tag with each nucleotide base.

16. The method as claimed in claim 1, wherein step (C) is performed.

17. The method as claimed in claim 1, further comprising the step of:

(F) sequencing the target nucleic acid molecule by identifying the magnifying tags associated with the target nucleic acid molecule.

18. The method as claimed in claim 17, wherein 2 or more nucleotide bases are sequenced per cycle.

19. The method as claimed in claim 18, wherein 4 or more nucleotide bases are sequenced per cycle.

20. The method as claimed in claim 17, wherein the signal associated with each nucleotide base is magnified by increasing the number of times that said nucleotide base appears in said sequence.

21. The method as claimed in claim 17, wherein the resulting magnified signal is converted into readable signals and said sequencing is carried out by assessing the readable signals.

22. The method as claimed in claim 17, wherein each readable signal comprises a pattern made up of a single signal event which creates a unique signal on each magnifying tag.

23. The method as claimed in claim 1, wherein said method is performed on a sample comprising a heterogeneous mixture of target nucleic acid molecules.

24. The method as claimed in claim 17, wherein said method is performed on a sample comprising a heterogeneous mixture of target nucleic acid molecules.