Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1998-12-03
2003-10-28
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C436S006000, C436S091000, C436S169000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06638760
ABSTRACT:
2. FIELD OF THE INVENTION
The present invention relates to methods and apparatuses for flow-through capture and optional recovery of nucleic acids.
3. BACKGROUND OF THE INVENTION
Nucleic acid hybridization, i.e., the ability of nucleic acid strands of complementary sequence to form duplexed hybrids, is one of the most powerful analytical techniques in the biological sciences. One of the most widely used hybridization techniques today is the “Southern blot” method discovered by Southern (Southern, 1975, J. Mol. Biol. 98:503-507). In this method, a target denatured DNA is immobilized on a filter or membrane, such as a nitrocellulose or nylon membrane. The membrane is then incubated in a buffer solution which contains a labeled oligonucleotide probe complementary to a region of the immobilized target DNA under conditions wherein the target and probe hybridize. Following wash steps, the presence or absence of hybridization is determined by detecting the label, with a positive detection indicating the presence of hybridization. The above method has also been used with immobilized RNA targets. When used with RNA the method is called “Northern blotting.”
While powerful methods, Southern and Northern blotting suffer from several drawbacks. First, the methods cannot be used to study multiple sequences simultaneously within the same membrane in a single run, i.e., without the time-consuming procedures of repeat hybridization by different probes. Second, available membranes are generally unable to provide high immobilization efficiencies for target nucleic acid fragments containing fewer than 100 bp. Third, the hybridization kinetics are slow; oftentimes several hours or even several days are required for the probe and target to form a hybridized complex. Lastly, the Southern and Northern techniques suffer from the drawback that the target nucleic acid cannot be efficiently eluded from the membranes for subsequent use.
The slow hybridization kinetics observed with the Southern and Northern methods are thought to be caused by three main factors. First, since the whole membrane must be covered with hybridization solution, the concentration of probe available for hybridizing to the immobilized target DNA or RNA is extremely low. Since hybridization kinetics are governed by a bimolecular collision process, the dilute probe concentration has an enormous effect on the rate by which the probe “finds” and hybridizes to the target DNA or RNA. Second, the majority of the probe solution does not contact the membrane during the incubation process. This lowers the effective probe concentration even further, and also increases the likelihood that, if the target was initially double-stranded, the target strands will re-anneal at a faster rate than hybridization will occur. Third, a large proportion of the target nucleic acid is immobilized within the interior pores of the membrane, and is therefore inaccessible for hybridization. Thus, the hybridization kinetics are slowed even further by the probe having to diffuse into the pores of the membrane.
Recently, it has been postulated that hybridization can be used to sequence DNA or RNA. The sequencing by hybridization method (“SBH”), first described by Lysov et al. utilizes a set of short oligonucleotide probes of defined sequence to search for complementary sequences on a longer strand of target DNA or RNA. The hybridization pattern is then used to reconstruct the sequence of the target DNA or RNA (Lysov et al., 1988, Dokl. Acad. Nauk SSSR 303:1508-1511; see also, Bains & Smith, 1988, J. Theor. Biol. 135:303-307; Drmanac et al., 1989, Genomics 4:114-128; Strezoska et al., 1991, Proc. Natl. Acad. Sci. USA 88:10089-10093; Drmanac et al., 1993, Science 260:1649-1652).
Since the emergence of SBH, many new techniques for fabricating immobilized sets of probes have emerged. For example, Southern et al. constructed an array of 256 octanucleotides covalently attached to a glass plate using a solution-channeling device to direct the oligonucleotide probe synthesis (Southern et al., 1992, Genomics 13:1008-1017). Because the identity of the probe at each site is known, the entire array can be simultaneously hybridized with the target nucleic acid in a single assay; the hybridization pattern directly reveals the identities of all complementary probes.
In a similar vein, Pease et al. describe the use of photoprotected nucleoside phosphoramidites and light to direct the synthesis of a miniaturized array of 256 octanucleotides on a glass substrate in a spatially-addressable fashion (Pease et al., 1994, Proc. Natl. Acad. Sci. USA 91:5022-5026). The resulting miniaturized array measured 1.28×1.28 cm and took only 16 reaction cycles and 4 hours to synthesize. Like the array of Southern, the miniaturized array can be simultaneously hybridized with the target nucleic acid to reveal the identities of all complementary probes.
Dubiley et al. describe the use of oligonucleotide microchips that have been manufactured by immobilizing presynthesized oligonucleotides within polyacrylamide gel pads arranged on the surface of a microscope slide (Dubiley et al., 1997, Nucl. Acids Res. 25(12):2259-2265). The microchips have been applied to sequence analysis (Yershov et al., 1996, Proc. Natl. Acad. Sci. USA 93:4913-4918), mutation analysis (Drobyshev et al., 1997, Gene 188:45-52) and identification of microorganisms.
Hybridization with the above-described probe arrays provides at least two advantages over the Southern and Northern blotting techniques. First, since each probe is attached to a discrete site on the substrate, the target DNA or RNA can be assayed for its ability to form hybrids with a plurality of probes in a single experiment. Second, the hybridized complexes can be readily dissociated and the target nucleic acid recovered for subsequent use. However, since these methods also rely on immersion hybridization techniques, i.e., the entire substrate must be immersed in hybridization solution containing the target nucleic acid, the kinetics of hybridization are slow. Depending on the concentration and length of the target nucleic acid, the formation of hybridized complexes can take on the order of hours or even days. Moreover, the methods require a large volume of hybridization buffer, and hence quite a large quantity of target nucleic acid.
Due to the ability of nucleic acids to form duplexes with a high degree of specificity, hybridization has also been used to capture a target nucleic acid from a sample. Such methods can be used to determine whether the sample contains the target nucleic acid, to quantify the amount of target nucleic acid in the sample, or to isolate the target nucleic acid from a mixture of related or unrelated nucleic acids. To this end, capture polynucleotides capable of hybridizing to a target nucleic acid of interest have been immobilized on a variety of substrates and supports for use in capture assays.
For example, capture polynucleotides have been immobilized within the wells of standard 96-well microtiter plates (Rasmussen, et al., 1991, Anal. Chem. 198:138-142), activated dextran (Siddell, 1978, Eur. J. Biochem. 92:621-629), diazotized cellulose supports (Bunneman et al., 1982, Nucl. Acids Res. 10:7163-7180; Noyes and Stark, 1975, Cell 5:301-310) polystyrene matrices (Wolf et al., 1987, Nucl. Acids Res. 15:2911-2926) and glass (Maskos & Southern, 1992, Nucl. Acids Res. 20:261-266), to name a few. However, these systems suffer from very poor diffusion characteristics, leading to slow, inefficient hybridization.
In part to overcome the slow hybridization kinetics of available immobilization supports, the art has also attempted to immobilize capture polynucleotide on beads, including submicron latex particles (Wolf et al., 1987, supra), avidin-coated polystyrene beads (Urdea et al., 1987, Gene 61:253-264) and magnetic beads (Jakobson et al., 1990, Nucl. Acids Res. 18:3669). However, the beads are difficult to manipulate, particularly magnetic beads, which require elaborate isolation stations to retain the beads and precise liquid handling to a
Chen Jer-Kang
Chiesa Claudia
Fry George A.
Furniss Vergine C.
Lambert Stephen M.
Lu Frank
PE Corporation (NY)
Whisenant Ethan
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