Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
2000-06-27
2003-03-18
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S600000, C204S603000, C204S451000, C204S452000
Reexamination Certificate
active
06533914
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns the field of high throughput assays of molecules. More particularly, the present invention concerns methods and apparatus of use in DNA sequencing and other high throughput assays, using a novel hybrid apparatus comprising an array of capillaries attached to a microfabricated chip injector.
2. Description of Related Art
DNA sequencing chemistry was first developed by Sanger et al. (1977) and by Maxam and Gilbert (1977). Sanger's dideoxy chain termination method is the most widely used for high-volume sequencing, due to the development of automated fluorescence sequencing based on labeled primers or terminators (Smith et al., 1986; Prober et al., 1987; Tabor et al., 1990; Ansorge et al., 1987). Implementation of this technology has produced automated slab-gel-based sequencers with 1-2 kb/hr capacity (Hunkapiller et al., 1991). This capacity may be pushed to 5 kb/hr through incremental improvement by increasing the number of lanes, decreasing the gel thickness, and improving the labeling chemistry and the detection capabilities. Such improvements will plateau unless revolutionary technique(s) are invented and applied. Generally, the slab gel format is not easily adapted for automated sample loading and it is probably incompatible with further efforts to miniaturize the sequencing process.
A number of advances have been made in DNA sequencing technology since 1990. These originate from two developments. First, by reducing the cross section of the gel cavity, higher electric fields can be applied without producing gel-heating anomalies, thereby providing faster separations. Second, laser-excited fluorescence detection is so sensitive that smaller separation lanes can be easily detected.
A number of workers have explored the use of capillary gel electrophoresis (CGE) for the rapid separation of DNA extension fragments in one-color and eventually 4-color detection formats (Drossman et al., 1990; Luckey et al., 1990; Swerdlow et al., 1991; Cohen et al., 1990; Ruiz-Martinez et al., 1993; Best et al., 1994). Capillary electrophoresis (CE) separations of DNA sequencing fragments are about 10 times faster than slab gels and are often complete in under 2 hr. However, the throughput of CE-based DNA sequencing has been limited by the lack of a method for running multiple capillaries (lanes) in parallel.
Mathies and coworkers (Huang et al., 1992a, 1992b; Mathies et al., 1992) developed a solution to this limitation by using a laser-excited confocal fluorescence scanner (Mathies et al., 1994) to interrogate bundled capillaries. The capillaries were translated past the focused laser beam and sequentially sampled. The capillaries separated at the injection end of the array to facilitate rapid sample loading from a microtitre dish array.
Several different groups have developed alternative capillary array apparatus. Kambara and coworkers (Takahashi et al., 1994) developed a capillary array system based on sheath flow detection scheme. Two different laser excitation beams were passed through the sheath flow and the fluorescence was imaged to a CCD for multicolor detection. This format has the advantage that all lanes are continuously excited and the flow cell has good optical quality with low scattering noise. Disadvantages of this arrangement include the complexity of the sheath flow cell and extra band-broadening due to the electric field distortion in the flow cell. A conceptually similar system was described by Dovichi et al. (1995). Ueno and Yeung (1994) developed a capillary array system where the laser is line-focused on a stationary array and the resulting fluorescence is imaged to a CCD for detection. A two-color-ratio method was used for DNA sequencing. Two 96-capillary-array DNA sequencers have been successfully developed and are currently used. One is based on Mathies' confocal detection design and the other is based on Kambara's sheath flow arrangement.
Research efforts have also been given to sequencing with short separation channels on microfabricated CE chips (Woolley et al., 1995; Schmalzing et al., 1998; Liu et al., 1999) or short capillaries (Muller et al., 1998) to increase the separation speed. Woolley and Mathies (1995) performed high-speed separations of DNA sequencing fragments on microfabricated CE chips. DNA separations were achieved in 50 &mgr;m×8 &mgr;m×3.5 cm channels microfabricated in a 2-in.×3 in. glass sandwich structure. Approximately 150 bases of four-color sequencing mixture were separated in 9 min and base-assigned with an accuracy of 97%. Single-base resolution was obtained out to 200 bases for both the one- and four-color separations. Alternative methods for detection of separated molecules on CE chips have been disclosed (U.S. Pat. No. 5,906,723, incorporated herein by reference in its entirety.)
Theoretically, high-speed separation should be achieved on capillaries provided they are short. Muller et al. (1998) have experimentally demonstrated this using a 7-cm-long (50-&mgr;m-i.d.) capillary and one-color sequencing mixture. Single base resolution was observed up to 300 bases in 3 min. However, it is difficult to arrange 96 or more such capillaries to carry out sample injection and separation for high-throughput DNA sequencing. Recently, Schmalzing, et al (1998) performed a separation of one-color sequencing mixture using an 11.5-cm-long microfabricated channel. Single-base resolution was obtained up to ~400 bases in 14 min. Liu et al. (1999) have demonstrated high-speed DNA sequencing on a 7 cm-long microfabricated channel. Single-base resolution of reached 500 bases in 9.2 min using a one-color sequencing mixture, and four-color sequencing exhibited a base-calling accuracy of 99.4% up to 500 bases.
Current efforts on the development of high-speed and high-throughput DNA sequencing are in two major areas, conventional CGE and microfabricated electrophoresis chips. Research with conventional CGE focuses on improving the sieving matrix and separation conditions, and increasing the number of capillaries. Research on microfabricated electrophoresis chips is more exploratory. In order to achieve high-speed and high-throughput analysis, Mathies et al. (1999) developed a radial chip. This chip has a common anode reservoir in the center of a circular 10 cm diameter wafer and an array of 96 channels extending outward toward injector units at the perimeter of the wafer. A rotary scanning detection system consists of a rotating objective head coupled to a four-color confocal detection unit. High-speed and high-throughput assays have been demonstrated on this radial chip for genotyping. High quality sequencing has not been obtained due to the limited effective separation distance.
Other attempts are directed to the manufacture of large “chips” in order to achieve high quality sequencing separation. These approaches gain back the read-length from conventional CGE, but give up the separation speed of microfabricated CE chips. In addition, “micro” fabrication of a half-meter size chip without defects is challenging.
Sequencing separation using short separation channels (Schmalzing et al., 1998; Liu et al., 1999) improves separation speed about 10 fold compared to conventional capillaries (~40 cm). However, the sequencing read-lengths diminish by a factor of 1.5 to 2.
An unresolved need exists in the art for the development of high speed, high-throughput DNA sequencing methods and apparatus that are capable of reading DNA sequences significantly longer than 500 bases, using small amounts of DNA sample in a small sample volume. None of the methods or apparatus discussed above are capable of such separations.
SUMMARY OF THE INVENTION
The present invention solves a long-standing need in the art by providing a hybrid apparatus for high-speed, high throughput and long read length DNA sequencing separation, comprising a microfabricated chip injector attached to an array of one or more capillaries. Within the scope of the invention almost any number of capillari
Blakely & Sokoloff, Taylor & Zafman
Brown Jennine
Nakashima Richard A.
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