SINGLE MOLECULE ANALYSIS WITH HIGH ACCURACY

Abstract

The invention relates to a process for analysing single molecules, in particular for sequencing of single nucleic acid molecules.

Claims

1. A device for analyzing at least one individual single molecule, comprising: (a) a support for positioning a single molecule to be analyzed on an individual spot, comprising at least one individual sample spot, wherein said spot has a diameter in the range of about 1 nm-20 nm and the distance between individual spots is at least about 5 times the diameter of the spot, (b) a light source which provides at least one individual illuminated volume element at an individual sample spot on the support, for illuminating a single molecule at an individual spot, (c) a light detector which comprises at least one detection pixel wherein said detection pixel has a diameter in the range of about 0.5 μm-50 μm and the distance between said detection pixels is at least about 2 times the diameter of the detection pixel, for individually detecting light emitted from a single molecule at an individual spot, (d) means for optically projecting said detection pixel onto the support, comprising an optical pathway between the light source, the support and the light detector, wherein the optical projection has a diameter in the range of about 100 nm-5 μm and is about 10-200 times smaller than the size of the detection pixel on the detector, (e) means for aligning an individual sample spot on the support to the optical projection of a single detection pixel such that the center of the individual sample spot is aligned to the projection of the single detection pixel on the support, wherein the distance between individual sample spots on the support is equivalent to the distance between optical projections of detection pixels on the support, and (f) means for detecting the signals from individual detection pixels, wherein said signals result from an event associated with a single molecule positioned on an individual spot.

2. The device according to claim 1, wherein the distance between individual sample spots on the support is from 5 nm-5000 nm.

3. The device according to claim 1, wherein said event associated with a single molecule positioned on an individual spot is caused by association or dissociation of a labeling group or a time dependent change of light emission.

4. The device according to claim 1, wherein said device has an accuracy of at least 99.0% with a single measurement.

5. The device according to claim 4, wherein said device has an accuracy of 99.9% with a single measurement.

6. The device of claim 1, wherein the light detector detects light emitted from single molecules comprising detectable labelling groups.

7. The device of claim 1, wherein said single molecule is a single nucleic acid molecule.

8. The device of claim 1, wherein said at least one sample spot further comprises a) a nucleic acid-synthesizing enzyme molecule or a nucleic acid degrading enzyme molecule, and b) fluorescence labelled nucleotide building blocks in free form and/or incorporated into a nucleic acid molecule.

9. The device of claim 7, wherein the single nucleic acid molecule is circular or linear.

10. The device of claim 8, wherein the nucleic acid-synthesizing enzyme molecule or the nucleic acid-degrading enzyme molecule is immobilized on said sample spot.

11. The device of claim 1, wherein the support has a planar surface.

12. The device of claim 1, wherein the support has a surface selected from the group consisting of glass, plastic, metal, quartz, semi-metal, metal oxide and a composite comprising a plurality thereof.

13. The device of claim 1, wherein said at least one sample spot comprises a coated surface area on the support or a particle deposited on the surface of the support.

14. The device of claim 13, wherein the surface of said sample spot and/or of a particle deposited thereon is a metal selected from the group consisting of Au, Ag, Cr, Ni and Al, a semi-metal and a silane.

15. The device of claim 13, wherein the surface of a sample spot and/or of a particle deposited thereon is modified with a capturing reagent selected from the group consisting of biotin, streptavidin and another high-affinity capturing reagent.

16. The device of claim 1, wherein the light detector is selected from the group consisting of a multipoint single photon avalanche detector (SPAD), a CCD camera and a CMOS detector matrix.

17. The device of claim 1, wherein the light detector comprises 10.sup.3 to 10.sup.6 individual detection pixels.

18. The device of claim 1, wherein the position of a sample spot relative to a detection pixel is aligned by an adjustment element.

19. The device of claim 1, wherein the light source is a multipoint laser.

20. The device of claim 1, wherein the at least one individual illuminated volume element is provided by a diffractive optical element.

21. The device of claim 20, wherein the diffractive optical element is introduced into the optical pathway in a total internal reflection (TIR) setup.

22. The device according to claim 1, wherein said device comprises sample spots sufficient for analyzing a population of sequences.

23. The device of claim 22, wherein the population of sequences comprises at least 10, at least 10.sup.2, at least 10.sup.3, or at least 10.sup.4 individual members.

24. The device according to claim 1, wherein the distance between individual spots is 20-500 times the diameter of the spots.

25. A device for analyzing single molecules, comprising: (a) a support comprising a plurality of individual sample spots, wherein said spots have a diameter in the range of about 1-20 nm and the distance between individual spots is at least about 5 times the diameter of the spot, for positioning a single molecule to be analyzed on an individual spot, (b) a light source which provides a plurality of individual illuminated volume elements at the spots on the support, for illuminating single molecules at individual spots, (c) a light detector which comprises a plurality of detection pixels, wherein said detection pixels on the detector have a diameter the range of about 0.5 μm-50 μm, wherein the distance between said detection pixels is at least about 2 times the diameter of the detection pixel, for individually detecting light emitted from a single molecule at an individual spot, (d) a means for optically projecting said detection pixels onto the support comprising an optical pathway between the light source, the support and the light detector, wherein the optical projection has a diameter in the range of about 100 nm-5 μm and is about 10-200 times smaller than the size of the detection pixel on the detector, (e) means for aligning each individual sample spot on the support to the optical projection of a single detection pixel such that the center of the individual sample spot is aligned to the projection of the single detection pixel on the support, wherein the distance between individual sample spots on the support is equivalent to the distance between optical projections of detection pixels on the support, and (f) a means for detecting light from individual detection pixels wherein said light results from an event associated with a single molecule positioned on an individual spot.

Description

[0139] Furthermore, the figures below are intended to illustrate the present invention.

[0140] FIG. 1 shows a laser point matrix with confocal illuminated volume elements generated by a diffractive optical element as described on WO 2002/097406.

[0141] FIG. 2 shows a 1024-pixel SPAD detector. SPAD detectors combine high sensitivity over a broad spectral range of e.g. 350-900 nm with high-time resolution (≤1 ns).

[0142] The pixel number of a detector is preferably about 100 to about 500.000. The pixel diameter on the detector is preferably about 1-20 μm and the detector pitch length (i.e. the distance between individual pixels on the detector) is about 2-200 μm.

[0143] In the following, Table 2 shows preferred pixel diameter and pitch length sizes on the detector.

TABLE-US-00002 TABLE 2 Pixel no. 1024 4096 16384 409600 Pixel diameter (μm) 20 10 5 1 Pitch length (μm) 100 50 25 5

[0144] FIG. 3 shows alignment of sample spots (black) in the center of optical projections of detection pixels (grey) on the support.

[0145] Table 3 demonstrates the relationship of sample spot diameter (SD), pixel projection diameter (PD) and sequencing accuracy (A). Further sample spot distance which is equivalent to pixel projection distance (projection pitch) and pixel magnification (ratio of pixel diameter to pixel projection diameter) are shown. The sequencing accuracy may be calculated as follows: A=exp−(SD/PD).sup.2

TABLE-US-00003 TABLE 3 Pixel Sample spot projection Sample spot diameter diameter distance Accuracy SD (nm) PD (nm) (nm) A 100× Magn. 10 200 1000 0.9975 60×. Magn. 10 333 1665 0.9990

[0146] Alignment of detector pixel matrix and sample spot matrix is preferably carried out by a nanometer precision x-y piezo-adjustment in a detector-driven feedback loop (Physik Instrumente GmbH & Co. KG, Karlsruhe, Germany).

[0147] FIG. 4 shows an objective type of internal reflection (TIR) set up with laser based excitation. A collimated laser beam is focussed in the back focus plane of a high NA objective. The collimated laser beam is totally reflected by the glass surface of the illuminated area. Fluorescent molecules at the surface are excited to fluorescence in the evanescent field which decays exponentially with the distance from the surface. With introduction of a diffractive optical element after the lens focussing the collimated laser beam a multiplicity of laser beams can be generated which are reflected by TIR. Together with the pixels of the SPAD matrix and their projection on the TIR surface a multitude of confocal volume elements can be generated.

[0148] Abbr. OL objective lens, BFP back focal plan, DM dichroic mirror, DOE diffractive optical element, GC angular beam control, EMF emission filter, SPAD single photon avalanche diode matrix.

[0149] FIG. 5 shows that a TIR(F) setup allows adjustment of the angle of the incoming and totally reflected beam and hence the size of the evanescence field. A volume of a few zeptoliters can be reached particularly with the use of DOE split laser beams. The advantage of a flat surface is the application of free flow on the surface and enzymatic operation on unkinked DNA strands.

[0150] FIG. 6: The primary accuracy, p, is shown together with the probability of correct read out of one base when both strands of a DNA are measured.