Detection of nucleic acid polymorphisms

• US 20040072200A1
• Filed: 11/25/2003
• Published: 04/15/2004
• Est. Priority Date: 11/13/2000
• Status: Abandoned Application

• Current Assignee: Gnothis Holding SA
• Original Assignee: Gnothis Holding SA
• Inventors: Edman, Lars, Foeldes-Papp, Zeno, Rigler, Rudolf
• Application Number:
  US10/416,574
• Publication Number:
  US 20040072200A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2537/163 blocking probe
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads

Abstract

Methods are described methods functioning at the single-molecule level for characterizing nucleotide polymorphisms.

Claims

1. A method for characterizing nucleic acid polymorphisms, including the steps:

(a) provision of a nucleic acid template to be investigated,

(b) annealing of at least one starting primer onto the nucleic acid template, with the 3′

end of the starting primer being located upstream of a nucleic acid polymorphism to be investigated,

(c) extension of the starting primer with at least one fluorescence-labeled nucleotide and

(d) detection of nucleotides incorporated into the starting primer by single-molecule determination.

2. The method as claimed in claim 1, characterized in that the detection of incorporated nucleotides includes a separation of the extended starting primer from unincorporated nucleotides.

3. The method as claimed in claim 1 or 2, characterized in that the detection of the incorporated nucleotide includes determination of at least part of the sequence of the extended starting primer.

4. The method as claimed in any of the preceding claims, characterized in that to carry out the single-molecule determination the starting primer is coupled to a carrier particle.

5. The method as claimed in claim 3 or 4, characterized in that the single-molecule sequence determination includes the steps:

(a) introduction of the carrier particle into a sequencing device including a microchannel,

(b) retention of the carrier particle in the sequencing device,

(c) progressive elimination of individual nucleotide units from the immobilized nucleic acid molecule,

(d) at least partial determination of the base sequence of the nucleic acid molecule on the basis of the sequence of the eliminated nucleotide units.

6. The method as claimed in any of the preceding claims, characterized in that an enzymatic elimination of nucleotides attached to the starting primer is effected by an exonuclease, in particular by T7 DNA polymerase as exonuclease, E. coli exonuclease I or E. coli exonuclease III.

7. The method as claimed in any of the preceding claims, characterized in that a single starting primer is used.

8. The method as claimed in any of claims 1 to 6, characterized in that a plurality of starting primers is used.

9. The method as claimed in either of claims 7 or 8, characterized in that the extension reaction includes the attachment of a single fluorescence-labeled chain-termination molecule.

10. The method as claimed in claim 9, characterized in that the chain-termination molecule is a dideoxynucleotide.

11. The method as claimed in either of claims 7 or 8, characterized in that the extension reaction includes the attachment of a plurality of fluorescence-labeled nucleic acid units.

12. The method as claimed in claim 11, characterized in that one or more pairs of starting and blocking primers are employed, where the 5′

end of each blocking primer binds to the nucleic acid template at a predetermined distance downstream of the 3′

end of the relevant starting primer, with the 3′

end of the blocking primer being blocked.

13. The method as claimed in claim 12, characterized in that a plurality of pairs of starting and blocking primers is employed, and the starting/blocking primer pairs can be identified by means of different codings.

14. The method as claimed in claim 12 or 13, characterized in that the blocking of the 3′

end of the blocking primer is reversible, where appropriate with the exception of the blocking primer which binds furthest downstream.

15. The method as claimed in any of claims 12 to 14, characterized in that blocking primers carrying a 3′

-phosphate group are used.

16. The method as claimed in any of claims 12 to 15, characterized in that a covalent bond is formed between the starting primer extended by fluorescent nucleotides, and the blocking primer.

17. The method as claimed in any of claims 12 to 16, characterized in that the covalent bond is formed enzymatically, for example by using a ligase.

18. The method as claimed in claim 16 or 17, characterized in that at least one blocking primer carries a 5′

-phosphate group.

19. The method as claimed in any of claims 14 to 18, characterized in that the gap(s) between the starting primers extended by fluorescent nucleotides, and the blocking primers located downstream in each case, after removal of the 3′

blocking of the blocking primer, are filled in by deoxyribonucleotides, and covalent bonds are formed between the extended blocking primers and the starting primers located directly downstream.

25. The method as claimed in any of the preceding claims, characterized in that a carrier particle made of plastic, glass, quartz, metals, metalloids, metal oxides or of a composite material is used.

26. The method as claimed in any of the preceding claims, characterized in that the carrier particle has a diameter of from 1 nm to 10 μ

m.

27. The method as claimed in any of the preceding claims, characterized in that the the nucleic acid matrix is immobilized on the carrier particle via a 5′

terminus or the starting primer is immobilized via its 3′

terminus by means of bioaffinity interactions.

28. The method as claimed in claim 1, characterized in that a biotinylated nucleic acid molecule is immobilized on an avidin- or streptavidin-coated carrier particle.

29. The method as claimed in any of the preceding claims, characterized in that the different fluorescent labels are distinguished on the basis of the wavelength, the lifetime of the excited state or a combination thereof.

30. The method as claimed in any of the preceding claims, characterized in that the determination takes place by confocal single-molecule detection or/and by time-resolved decay measurement.

31. The method as claimed in any of the preceding claims, characterized in that the determination includes the measurement of a cross-correlated signal which originates from a nucleic acid molecule, or nucleic acid molecule complex, comprising at least two different labels, especially fluorescent labels.

20. A method for characterizing nucleic acid polymorphisms in a microwell, including the steps:

(a) provision of a carrier particle with a nucleic acid molecule which is immobilized thereon and consists of a single-stranded nucleic acid template and of a starting primer,

(b) extension of the starting primer by a fluorescence-labeled chain-termination molecule,

(c) where appropriate washing of the well to remove unincorporated labels and

(d) detection of the fluorescent label incorporated into the starting primer.

21. The method as claimed in claim 20, characterized in that a semiconductor laser or/and semiconductor detector integrated into the microwell is used for the excitation or/and the detection of the fluorescence.

22. The method as claimed in claim 20 or 21, characterized in that a plurality of reactions is carried out in parallel or sequentially on one microwell plate.

23. The method as claimed in any of claims 20 to 22, characterized in that only one type of labeled nucleotide selected from the group consisting of ddATP, ddUTP, ddTTP, ddCTP, ddGTP is available for the extension of the starting primer.

24. The method as claimed in any of claims 20 to 22, characterized in that a plurality of chain-termination molecules which can be distinguished by their fluorescent label is available for the extension of the starting primer.

32. A method for increasing the detection efficiency in the detection of the fluorescence from single molecules, characterized in that a dispersion element is used to separate the light of different wavelengths.

33. The method as claimed in claim 32, characterized in that a prism or grating is used as dispersion element.