Multiplexed capillary electrophoresis system

Claims

What is claimed is:

1. A multiplexed capillary electrophoresis system comprising;

(a) a capillary array of coplanar parallel capillaries, each capillary having an interior portion for placement of a target species, and an annular wall with a first transparent portion defining a transparent path extending through the capillary array perpendicular to the capillaries;

(b) a coherent light source positioned to direct a beam of coherent light along the transparent path and through the interior portion of each capillary to induce emission from the target species;

(c) a filter positioned to split the target species emission into first and second emission channels; and

(d) an image array detector positioned to detect the target species emission.

2. The system of claim 1 wherein the first transparent portion extends around the capillary.

3. The system of claim 1 wherein the transparent path comprises a plane extending through the capillaries.

4. The system of claim 1 comprising a capillary array of at least about 100 coplanar parallel capillaries.

5. The system of claim 1 wherein the first transparent portion is transparent to light having a wavelength about equal to the wavelength of the beam of coherent light.

6. The system of claim 1 wherein the first transparent portion is transparent to light having a wavelength of about 200-1500 nm.

7. The system of claim 1 wherein the parallel capillaries are substantially adjacent to each other.

8. The system of claim 1 wherein the first transparent portion is surrounded by a medium having a refractive index of about 1.3-1.5.

9. The system of claim 1 wherein the capillary array is immersed in water.

10. The system of claim 1 wherein a collimating focusing lens is interposed between the coherent light source and the capillary array.

11. The system of claim 1 wherein the beam of coherent light has a diameter of less than about 300 .mu.m in the capillaries.

12. The system of claim 11 wherein the beam of coherent light has a diameter of less than about 75 .mu.m in the capillaries.

13. The system of claim 1 wherein a beam expander is used to expand the beam of coherent light perpendicular to the capillary array.

14. The system of claim 1 wherein the coherent light source comprises a laser having a power output of about 0.5-50 mW.

15. The system of claim 1 wherein the first transparent portion exhibits substantially no fluorescence when exposed to the beam of coherent light.

16. The system of claim 1 wherein each annular wall has a second transparent portion for optically coupling the transparent path to a location external to the capillary array.

17. The system of claim 16 wherein the location external to the capillary array comprises a planar surface parallel to the capillary array.

18. The system of claim 16 wherein the second transparent portion of each annular wall is contiguous with the first transparent portion of the annular wall.

19. The system of claim 16 wherein the location external to the capillary array contains an optical detector.

20. The system of claim 19 wherein the optical detector is a two-dimensional image array detector.

21. The system of claim 20 wherein the optical detector is selected from a group consisting of a charge-coupled device (CCD) and a charge-injection device (CID).

22. The system of claim 19 wherein at least one capillary is in fluid communication with a sample containing a fluorescent target species so that the sample is drawn into the capillary, and wherein the optical detector is capable of detecting fluorescence emission from the target species.

23. The system of claim 1 further comprising at least one optical fiber optically coupled to the transparent path.

24. A capillary electrophoresis system comprising:

a capillary array having a plurality of coplanar parallel capillaries, each capillary having an annular wall defining an interior portion, each annular wall having a transparent portion for optically coupling the interior portion to an image array detector;

(b) a coherent light source positioned to direct a single beam of coherent light into the interior portion of each capillary;

(c) an image array detector having linearly aligned pixels located in a plane parallel to the capillary array such that at least one of the capillaries is optically coupled to a plurality of pixels; and

a filter interposed between the image array detector and the capillary array.

25. The system of claim 24 wherein at least one capillary has at least one side wall proximate to the interior portion such that at least one pixel is optically coupled to the at least one side wall.

26. The system of claim 24 wherein the at least one capillary has first and second side walls on opposite sides of the interior portion and the less than about six pixels comprises a group of pixels having a leading pixel, a middle group of pixels, and a trailing pixel, such that the leading pixel is optically coupled to the first side wall, the middle group of pixels is optically coupled to the interior portion, and the trailing pixel is optically coupled to the second side wall.

27. The system of claim 26 wherein the middle group of pixels comprises two pixels.

28. The system of claim 26 wherein the middle group of pixels comprises one pixel.

29. The system of claim 24 further comprising an imaging lens for optically coupling the at least one capillary to the at least about six pixels.

30. The system of claim 24 wherein the image array detector is a two-dimensional image array detector.

31. The system of claim 30 wherein the two-dimensional image array detector is selected from a group consisting of a charge-coupled device (CCD) and a charge-injection device (CID).

32. The system of claim 30 wherein the two-dimensional image array detector is a charge-injection device (CID).

33. The system of claim 24 wherein the parallel capillaries are substantially adjacent to each other.

34. The system of claim 24 wherein when a target species is placed in the interior portion of each capillary and emission is induced by the coherent light, the filter is positioned to filter only a portion of the induced emission.

35. A capillary electrophoresis system comprising:

(a) at least one capillary having an annular wall defining an interior portion for placement of a fluorescent target species wherein said annular wall has a transparent portion for optically coupling the interior portion to a detector;

(b) a coherent light sourer positioned to direct a single beam of coherent light having a wavelength of about 200-1500 nm so as to contact the interior portion and induce fluorescence emission from the target species;

(c) first and second long-pass filters positioned to split the fluorescence emission into first and second emission channels, respectively; and

(d) a detector for simultaneously detecting the fluorescence emission in the first and second emission channels.

36. The system of claim 35 wherein the transparent portion extends around the capillary.

37. The system of claim 36 wherein the first long-pass filter is interposed between the detector and the transparent portion of an annular wall such that fluorescence emission passes through the first filter, and the second long-pass filter is interposed between the first filter and the transparent portion of the annular wall at an angle of about 1.degree.-89.degree. relative to the first filter, such that a portion of the fluorescence emission passes through the second filter before passing through the first filter.

38. The system of claim 37 wherein the angle is about 20.degree.-40.degree..

39. The system of claim 37 wherein the portion of fluorescence passing through both the first filter and the second filter is about 25%-75% of the fluorescence emission passing through the first filter.

40. The system of claim 36 wherein the second long-pass filter is interposed between the detector and the transparent portion of an annular wall such that fluorescence emission passes through the second filter, and the first long-pass filter is interposed between the second filter and the transparent portion of the annular wall at an angle of between about 1.degree.-89.degree. relative to the second filter, such that a portion of the fluorescence emission passes through the first filter before passing through the second filter.

41. The system of claim 35 wherein at least one capillary is in fluid communication with a sample containing a fluorescent target species such that the sample is drawn into the capillary and brought into contact with the beam of coherent light.

42. The system of claim 35 wherein the at least one capillary comprises a capillary array of coplanar parallel capillaries, the annular wall of each capillary having a first transparent portion defining a transparent path extending through the capillary array perpendicular to the capillaries, and wherein the coherent light source comprises a coherent light source positioned to direct the single beam of coherent light along the transparent path.

43. The system of claim 42 wherein each annular wall has a second transparent portion for optically coupling the transparent path to the detector.

44. The system of claim 43 wherein the detector comprises a first linear array detector for detecting fluorescence emission in the first emission channel and a second linear array detector for detecting fluorescence emission in the second emission channel.

45. The system of claim 43 wherein the detector comprises a two dimensional image array detector.

46. The system of claim 45 wherein the detector is selected from a group consisting of a charge-coupled device (CCD) and a charge-injection device (CID).

47. The system of claim 35 wherein the first long-pass filter has a wavelength cutoff value such that it transmits less than about 0.1% of light having a wavelength about equal to the wavelength of the beam of coherent light, and wherein the second long-pass filter has a wavelength cutoff value higher than the wavelength cutoff value of the first long-pass filter.

48. The system of claim 35 wherein the first long-pass filter comprises a Raman long-pass filter having a wavelength cutoff value about equal to the wavelength of the beam of coherent light.

49. The system of claim 35 wherein the single beam of coherent light has a wavelength of about 488 nm, and wherein the first long-pass filter is a Raman long-pass filter having a wavelength cutoff value of 488 nm and the second long-pass filter is a standard long-pass filter having a wavelength cutoff value higher than the wavelength cutoff value of the first long-pass filter.

BACKGROUND OF THE INVENTION

The use of capillary electrophoresis (CE) has greatly improved DNA sequencing rates compared to conventional slab gel electrophoresis. Part of the improvement in speed, however, has been offset by the loss of the ability (inherent in slab gels) to accommodate multiple lanes in a single run. Highly multiplexed capillary electrophoresis, by making possible hundreds or even thousands of parallel sequencing runs, represents an attractive approach to overcoming the current throughput limitations of existing DNA sequencing instrumentation.

Excitation and Detection Geometry. Various excitation and detection systems have been developed to accommodate parallel arrays in capillary electrophoresis. Laser-induced fluorescence (LIF) detection has been the major method employed in the automation of DNA sequencing. The incident laser beam and the collected fluorescence light are typically perpendicular to each other in order to reduce background noise due to light scattering. On-column excitation and detection are generally performed from above the parallel array through transparent windows formed in the capillaries. For example, in one system a beam expander and a cylindrical lens are used to distribute the laser light into a thin line that intersects the axes of the capillaries, which are mounted in a grooved block so as to reduce cross-talk (K. Ueno et al., Anal. Chem., 66, 1424 (1994)). Although a low detection limit and uniform distribution of excitation intensities can be achieved with this system, a long laser line compared to the array width has to be used due to the Gaussian intensity distribution. Thus, half of the laser light in the array region is wasted due to the longer laser line and the presence of the spacer grooves. Cross-talk, though manageable, is still in the range of 10% of the observed signal.

On-column detection has also been carried out using axial-beam laser-induced fluorescence detection by inserting optical fibers into an end of each separation capillary (J. A. Taylor et al., Anal. Chem., 65, 956 (1993)). However, the intrusion of optical fibers into the separation capillaries affects the electroosmotic flow and increases the possibility for contamination and clogging. Furthermore, the detection limit is higher.

A type of side-entry excitation in a single capillary system has also been reported (R. N. Zare et al., U.S. Pat. No. 4,675,300 (1987)). In that system, an optical fiber is used to deliver coherent light to a translucent portion of a capillary, and fluorescence is detected through the translucent portion using a second optical fiber positioned perpendicular to the first optical fiber. This method suffers from excess stray light contamination and lower collimation efficiency.

Increased laser power is generally advantageous in providing a larger analyte signal. However, fluorophores are easily bleached, i.e., their fluorescing characteristic is destroyed by the laser beam, even at the milliwatt level, negating any increase in excitation intensity. Thus an LIF geometry that produces high resolution analyte signals while using a lower power laser (i.e., less than 50 mW) would represent a needed improvement in the art.

Detection Methods and Devices. Highly multiplexed CE imposes great demands on the detection system. For example, in one approach, a two-color confocal fluorescence scanner is employed for 25 capillaries (X. C. Huang et al., Anal. Chem., 64, 967 (1992)). A mechanical stage is used to translate the capillary array across the optical region. Since data acquisition is sequential and not truly parallel, its use for hundreds of capillaries is limited. To be compatible with the high speed provided by CE and the high throughput of a large capillary array, a fast, sensitive, image array detector is required.

Recently, charge-coupled devices (CCDs) have been used as two-dimensional (n.times.m) image array detectors to pursue high-speed, high-throughput DNA sequencing. For example, a multiple sheath-flow apparatus and four-color detection system are used by S. Takahashi et al. (Anal. Chem., 66, 1021 (1994)). Two laser beams are combined into one to cross the flow streams in an array of 20 capillaries in a line for excitation, and a CCD is used for simultaneous detection perpendicular to the excitation beam. Superior stray-light rejection can be achieved with this system. However, many challenges remain in scaling up from 20 to hundreds or thousands of capillaries. Misalignment of individual sheath flows, turbulence in the flow paths, improper matching of the laser beam waist over a long distance with the core diameters containing the eluted fragments, and the possible need to incorporate an extra space between the capillaries to accommodate the sheath flow are just a few of the problems associated with scale-up. Moreover, CCD detectors make major data analysis and storage demands on a system. CCDs read one array row at a time, and the time spent reading any particular row cannot be lengthened or shortened as desired in response to the amount of information in that row. A two-dimensional image array detection system that allowed random addressing and variable exposure times would significantly reduce data storage and analysis demands, and save considerable amounts of time as well.

Nucleotide Identification in DNA Sequencing Experiments--"Base Calling". It is unlikely that capillary electrophoresis will ever provide migration times that are reproducible enough among a group of capillaries to allow running four sets of fragments generated from a single DNA sample in a DNA sequencing analysis (one set of fragments for each for nucleotide bases A,T,C, and G) in separate capillaries. Thus, methods have been developed to distinguish the four bases run on a single capillary. The one-color, four-intensity scheme is least desirable because of difficulties in controlling the polymerase and maximizing the signal-to-noise ratio (S/N) (H. Swerdlow et al., Anal. Chem., 63, 2835-2841 (1991)). The two-color, two-intensity scheme provides the advantages of a simpler optical arrangement, good light collection, and a straightforward algorithm (R. A. Mathies et al., Anal. Chem., 64, 2149-2154 (1992); D. Chen et al., Nucl. Acids Res., 20, 4873-4880 (1992)). However, like the one-color, four-intensity scheme, this scheme also assumes uniform incorporation of label by the polymerase which is often an incorrect assumption.

The technology in most common use is therefore still the four-color scheme originally reported by F. Sanger et al. (Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467 (1977)). Many optical arrangements have been developed for base calling with four-dye labels (S. Carson et al., Anal. Chem., 65, 3219-3226 (1993); R. Tomisaki et al., Anal. Sci., 10, 817-820 (1994); A. E. Karger et al., Nucl. Acids Res., 19, 4955-4962 (1991)). The four standard dyes (FAM and JOE, which are fluorescein derivatives, and ROX and TAMRA, which are rhodamine derivatives, available as the PRISM dyes from ABD division of Perkin Elmer, Foster City, Calif.) are by no means spectrally distinct, either in excitation or in emission. Currently available commercial instruments therefore use fairly narrow interference filters for emission and two laser wavelengths for excitation. Still, a complicated set of emmission ratios have to be employed for base