Design of capture molecules for the detection of amplicons with high sensitivity

• US 20060281112A1
• Filed: 05/17/2006
• Published: 12/14/2006
• Est. Priority Date: 03/24/2000
• Status: Abandoned Application

• Current Assignee: Eppendorf Array Technologies SA (Eppendorf Se)
• Original Assignee: Eppendorf Array Technologies SA (Eppendorf Se)
• Inventors: Remacle, Jose, Hamels, Sandrine
• Application Number:
  US11/436,379
• Publication Number:
  US 20060281112A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

The invention relates to a method for the design and/or the preparation of a polynucleotide capture molecule for detecting an amplicon having one strand serving as target to be detected and/or quantified after hybridization on said capture molecule, comprising the steps of: (a) selecting a primer pair defining the amplicon; (b) selecting a specific sequence of 10 to 100 nucleotides within the amplicon, such that said specific sequence defines two non-complementary ends of the amplicon; (c) defining the capture molecule having a capture portion that is complementary to the specific sequence selected in step b), and a spacer portion comprising at least 20 nucleotides; and (d) identifying among the two non-complementary ends of the amplicon a spacer end and a non-spacer end, respectively, such that the spacer end is non-complementary to the spacer portion of the capture molecule, and said spacer end exceeds said non-spacer end by at least 50 bases.

Claims

1. A method for designing a polynucleotide capture molecule for detecting an amplicon having one strand serving as target to be detected and/or quantified after hybridization on said capture molecule, said method comprising the steps of:

a) selecting a primer pair defining the amplicon;

b) selecting a specific sequence of 10 to 100 nucleotides within the amplicon, such that said specific sequence defines two non-complementary ends of the amplicon;

c) defining the capture molecule having a capture portion that is complementary to the specific sequence selected in step b), and a spacer portion comprising at least 20 nucleotides; and

d) identifying among the two non-complementary ends of the amplicon a spacer end and a non-spacer end, respectively, such that the spacer end is non-complementary to the spacer portion of the capture molecule, and said spacer end exceeds said non-spacer end by at least 50 bases.

2. A method for preparing a polynucleotide capture molecule for detecting an amplicon having one strand serving as target to be detected and/or quantified after hybridization on said capture molecule, said method comprising the steps of:

a) selecting a primer pair defining the amplicon;

b) selecting a specific sequence of 10 to 100 nucleotides within the amplicon, such that said specific sequence defines two non-complementary ends of the amplicon;

c) defining the capture molecule having a capture portion that is complementary to the specific sequence selected in step b), and a spacer portion comprising at least 20 nucleotides; and

d) identifying among the two non-complementary ends of the amplicon a spacer end and a non-spacer end, respectively, such that the spacer end is non-complementary to the spacer portion of the capture molecule, and said spacer end exceeds said non-spacer end by at least 50 bases.

3. A capture molecule obtainable by the method of claim 2.

4. The method for detecting an amplicon comprising the step of hybridizing the amplicon to the capture molecule of claim 3.

5. The method of claim 4 wherein the capture molecule is immobilized on a solid support such that the spacer portion is located between the solid support and the capture portion.

6. The method of claim 5 wherein the capture molecule is immobilized by its 5′

end.

7. The method of claim 5 wherein the capture molecule is immobilized by its 3′

end.

8. The method of claim 5 wherein the distal end of the spacer portion of the capture molecule has a nucleotide containing a free amino group.

9. The method of claim 4 wherein the amplicon has a length of at least 200 bp, preferably at least 300 bp, more preferably at least 400 bp.

10. The method of claim 4 wherein the amplicon has a length not exceeding 2000 bp.

11. The method of claim 5, wherein the spacer portion is a polynucleotide chain having a length of at least 40 nucleotides, preferably at least 90 nucleotides.

12. The method of claim 5, wherein the capture molecule comprises a spacer having at least 60% homology with SEQ ID NO:

2.

13. The method of any one of claims 4-12 wherein the capture molecule comprises a spacer having at least 60% homology with SEQ ID NO:

3.

14. The method of claim 5, wherein the capture portion contains from 10 to 100 nucleotides, preferably from 15 to 40 nucleotides, more preferably from 20 to 30 nucleotides.

15. The method of claim 5, wherein the density of capture molecules on the support is from 20 to 2000 fmoles/cm².

16. The method of claim 5, wherein capture molecules are immobilized in specifically localized areas of a solid support in the form of a micro-array of at least 4 capture molecules per cm², preferably at least 20 capture molecules per cm², more preferably at least 100 capture molecules per cm².

17. The method of claim 5, wherein the solid support is in the form of beads.

18. The method of claim 4, wherein at least two amplicons are detected with at least two capture molecules, and wherein the capture portions of said at least two capture molecules differ by at least 10%, preferably by at least 20%.

19. The method of claim 5, wherein the capture portion is capable of discriminating a target sequence from another sequence having less than 85% homology with the target.

20. The method of claim 4, comprising the use of a consensus primer pair capable of amplifying at least two target sequences having more than 60% homology, and comprising the use of capture molecules capable of detecting one each of the two target sequences.

21. The method of claim 4, wherein the amplicon is detected when present in a solution at a concentration of less than 10 nM.

22. The method of claim 4, wherein the amplicon is detected in real time as it is produced during a series of PCR cycles.

23. A kit for use in a method for detecting an amplicon, said kit comprising the capture molecule of claim 3.

24. The kit of claim 23, further comprising a solid support for immobilizing the capture molecule, said solid support being pretreated to react with the spacer portion of the capture molecule.

25. The kit of claim 23, further comprising a solid support having the capture molecule immobilized thereon.

26. The kit of claim 23, further comprising the means necessary for carrying out a hybridization of amplicons to the capture molecule.

27. The kit of claim 23 further comprising primer pairs and labeling means for carrying out PCR.