Identifying a target polynucleotide

• US 20090239213A1
• Filed: 10/12/2005
• Published: 09/24/2009
• Est. Priority Date: 10/13/2004
• Status: Abandoned Application

• Current Assignee: Lingvitae AS
• Original Assignee: Lingvitae AS
• Inventors: Lexow, Preben
• Application Number:
  US11/577,028
• Publication Number:
  US 20090239213A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals

Abstract

A method for identifying the presence of a single stranded target polynucleotide in a sample comprising the steps of (i) contacting the target polynucleotide, under hybridising conditions, with at least first and second polynucleotide probes, each of which comprise a first region complementary to adjacent non-overlappïng regions of the target polynucleotide; (ii) ligating together those first and second polynucleotides that hybridise to the target polynucleotide; (iii) optionally, amplifying any ligated polynucleotides; and (iv) determining whether the target polynucleotide is present in the original sample, by detecting any ligated polynucleotide, wherein at least one of the first and second polynucleotides comprise a second region having a defined polynucleotide sequence, with each individual nucleotide of the first region being represented by at least two nucleotides on the second region, and the ligated polynucleotide being identified by determining the second region of at least one of the first and second polynucleotide probes.

Claims

1. A method for identifying the presence of a single-stranded target polynucleotide in a sample, comprising the steps of:

(I) contacting the target polynucleotide, under hybridising conditions, 5 with at least first and second polynucleotide probes, each of which comprise a

first region complementary to adjacent non-overlapping regions of the target polynucleotide;

(ii) ligating together those first and second polynucleotides that hybridise to the target polynucleotide;

(iii) optionally, amplifying any ligated polynucleotides; and

(iv) determining whether the target polynucleotide is present in the original sample, by detecting any ligated polynucleotide, wherein at least one of the first and second polynucleotides comprise a second region having a defined polynucleotide sequence, with each individual nucleotide of the first region being represented by at least two nucleotides on the second region, and the ligated polynucleotide being identified by determining the second region of at least one of the first and second polynucleotide probes.

2. A method according to claim 1, wherein the first and second polynucleotide probes are DNA.

3. A method according to claim 1, wherein the first and second polynucleotide probes are RNA.

4. A method according to claim 1, wherein each of the bases A, T(U), G and C of the first region is represented by a combination of two sequential units in the second region, with each base represented by a different combination of the two units.

5. A method according to claim 1, wherein the first and second polynucleotide probes are labelled with a fluorescent moiety.

6. A method according to claim 1, wherein step (ii) comprises the addition of a ligase enzyme under conditions suitable for ligase activity.

7. A method according to claim 1, wherein the ligated polynucleotide of step (ii) is amplified in a polymerase reaction.

8. A method according to claim 7, wherein the polymerase reaction is quantitative and the amount of amplified polynucleotide is monitored.

9. A method according to claim 1, wherein the target polynucleotide is RNA.

10. A method according to claim 1, wherein the target polynucleotide is mRNA.

11. A method according to claim 1, wherein the target polynucleotide is DNA.

12. A method according to claim 1, for detecting a target polynucleotide that differs by one or more nucleotides compared to the wild-10 type sequence.

13. A method according to claim 12, for detecting a deletion, insertion or substitution.

14. A method according to claim 1, for detecting a single nucleotide polymorphism.

15. A method according to claim 1, for detecting a wild-type target sequence.

16. A method according to claim 1, wherein step (i) comprises the binding of a plurality of the same or different first and second polynucleotide probes to the target polynucleotide.

17. A method for identifying the order of at least two target sequences on a target polynucleotide, comprising:

(i) contacting the target polynucleotide, under hybridising conditions, with at least a first and second polynucleotide probe, each of which comprise a first region complementary to non-adjacent regions of the target polynucleotide;

(ii) performing a polymerase reaction to incorporate nucleotides between the at least two probes to thereby form a third polynucleotide; and

(iii) determining the order of, and distance between, the two target sequences on the target polynucleotide, by detecting any third polynucleotide,

wherein the first and second polynucleotides comprise a second region having a defined polynucleotide sequence, with each individual nucleotide of the first region being represented by at least two nucleotides on the second region, and

the order of and distance between the probes in the third polynucleotide being identified by detecting the second region of the first and second polynucleotide probes.