BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is further detailed with respect to the following nonlimiting figures. These figures depict only particular processes and apparatus according to the present invention with variants existing beyond those depicted.
FIG. 1A is a schematic of the concept of assembling of a nucleic acid microarray with external chamber providing space for hybridization of a nucleic acid target with a nucleic acid probe in solution;
FIG. 1B is a schematic denaturing of double-stranded oligonucleotides on the chip and electrophoretic transport of single-stranded DNA probes into the chamber with a nucleic acid target;
FIG. 1C is a schematic hybridizing a nucleic acid probe with target nucleic acids in solution;
FIG. 1D is a schematic transporting electrophoretically nucleic acid probes, which are not complementary to the target nucleic acid, back into a microarray chamber;
FIG. 1E is a schematic hybridizing free nucleic acid probes with DNA anti-probes of the same size immobilized on the microarray;
FIG. 2A is a schematic of an inventive assemblage of a DNA microarray with an external chamber for hybridization and with the labyrinth channel filled with solution having spots of immobilized DNA anti-probes on the bottom of the channel;
FIGS. 2B-2E are schematics of the operational steps for the assemblage of FIG. 2A where the steps so depicted are parallel to those of FIGS. 1B-1E, respectively;
FIG. 3 is a schematic depicting an operational mode for the assemblage of FIGS. 2A-2E in which a new array and new target nucleic acid are loaded into the respective chambers after an initial usage;
FIG. 4 is a schematic depicting an operational mode for the assemblage of FIGS. 2A-2E in which a new target nucleic acid is loaded into a target chamber and a recharged original array is provided after initial usage;
FIGS. 5A-5C are schematics of one mode of double-stranded nucleic acid denaturation through pH and electrode activation control;
FIG. 6A is a top view of a modular inventive assemblage well suited for manufacture to perform a process as depicted in FIGS. 1A-1E;
FIG. 6B is a central cross section through the assemblage of FIG. 6A;
FIGS. 7A-7E are cross-sectional schematics of a process of operating the assemblage of FIGS. 6A and 6B. FIG. 7A: manufacturer preloading of chambers with solutions, FIG. 7B: loading target nucleic acid sample into target chamber, FIG. 7C: electrophoretically transporting and hybridizing nucleic acid probes with target nucleic acid, FIG. 7D: electrophoretically returning nucleic acid probes not complementary to the target nucleic acid to the array chamber, FIG. 7E: hybridizing nucleic acid probes not complementary to the target nucleic acid to anti-probes immobilized and spotted in the array, and FIG. 7F: determination of double strand complex in a given spot by flow of washing and dye solutions;
FIG. 8 is a schematic of an alternate embodiment of an inventive assemblage having a target nucleic acid chamber in fluid communication with multiple probe-anti-probe arrays;
FIG. 9 are schematics depicting the connection of various inventive microarrays with chambers for hybridization in solution and with a channel for transporting nucleic acid probes by electrical field and detecting the probes passing through a detector;
FIG. 10 is a schematic depicting the two microarrays with a chamber for DNA hybridization in solution and with a channel for transporting nucleic acid probes by electrical field and detecting the probes passing through the detector;
FIG. 11 is a schematic depicting the connections of three DNA microarrays with a chamber for DNA hybridization in solution and with a channel for transporting along DNA probes by electrical field and detecting said DNA probes passing through the detector.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention has utility as a process and assemblage for identifying complementary sequences between a target nucleic acid and an array of nucleic acid probes initially forming a complex with an immobilized anti-probe nucleic acid sequence. A new process for signal detection on a DNA chip is provided in which the flow of charged nucleic acid probes released from anti-probe spots is determined by a detector as part of an inventive assemblage or an appended labyrinth based on the probe path and/or time of fight to a detector. According to the present invention, in addition to nucleic acid probes which are immobilized on a DNA microarray and nucleic acid targets which according to the prior art are free DNA molecules in solution, the present invention introduces an array of immobilized anti-probes of known sequence. The probes are selectively denatured from the anti-probes and brought into contact with a target nucleic acid under conditions in which hybridization between a nucleic acid probe and the target nucleic acid can occur of the probe nucleic acid sequence is complementary to that of the target nucleic acid. Thereafter, moving noncomplementary probes into contact with a series of immobilized complementary anti-probes under hybridization conditions, detection of those mobilized complementary anti-probes by various means that are not present as double-stranded complexes with nucleic acid probes indicates if a nucleic acid probe is complementary to the target nucleic acid. By separation of the target nucleic acid from the array of probe-anti-probes with a gel providing nucleic acid probe communication through electrophoretic movement, the target nucleic acid is provided in a variety of forms illustratively including free molecules in solution, as is known in the prior art, as well as immobilized on a solid surface, embedded in porous media such as a gel, adhered to particulate which is paramagnetic particles, semiconductor particles, metal particles or the like.
The present invention trough the inclusion of immobilized anti-probes capable of selectively being hybridized to nucleic acid probes that are amenable to transport affords the user multiple modes of operation with the resultant advantages illustratively including regeneration of the probe-anti-probe array, high throughput detection, time of flight detection, and combinations thereof. As a result, the present invention is amenable to a high level of manufacture so as to increase user throughput and provide target nucleic information consistent with that obtained from various types of prior art DNA chips. These uses illustratively include high throughput genotyping, resequencing, single nucleotide (SNP) genotyping, and gene expression chips. As a result, the present invention offers a degree of flexibility in operation, simplified manufacture and operation, and in regard to certain embodiments allows one to regenerate the inventive array for subsequent usage.
According to the present invention it is possible to prepare a complex of anti-probes and nucleic acid probe by first preparing a long double stranded nucleic acid which after treatment with specific restriction enzymes the second strand becomes a number of short nucleic acid strands hybridized to an elongated anti-probe strand. This procedure facilitates manufacture of numerous copies of nucleic acid probes by first amplifying long and repetitive double strand nucleic acid molecules and then treating such long double strand nucleic acid molecules with the appropriate restriction enzymes.
The present invention relies on a carrier capable of uniquely and reversibly binding a nucleic acid probe. In an inventive array, anti-probes are preferably isolated dimensionally in space or on a substrate. It is appreciated that in an array according to the present invention with anti-probes immobilized on a surface or within a porous matrix, nucleic acid probes can be harvested from a random mixture of short oligonucleotides, having a length of between 5 and 50 bases. Oligonucleotides harvested from the random mixture can be used as nucleic acid probes for subsequent hybridization and use in assays.
The arrangement of anti-probes in space so as to provide an inventive array includes a number of options in manufacture and operation. By way of example, anti-probes are coupled together to form an elongated strand. Preferably, the identity and position of each anti-probes along the strand is known. More preferably, spacer segments are provided intermediate between anti-probes along a strand so as to disfavor steric hindrance with probes pairing with the anti-probes sequences along the strand. It is appreciated that the specific inclusion of restriction sites within linker segments of the strand or knowledge as to such sites within anti-probes nucleic acid sequences provides for subsequent modification to replace a given anti-probes with a new anti-probe having different specificity. The ability to produce an elongated strand of anti-probes secured to a substrate by one or more strand termini creates an interaction environment with a probe in solution that is largely free of substrate surface interaction and the hindrances to probe-anti-probe complexation associated with a monolayer of probes immobilized on a substrate spot as in a conventional DNA microarray.
The operation of an inventive assemblage is illustrated in FIGS. 1A-1F. Throughout FIG. 1 like numerals are used throughout to depict the movement and status of various components throughout the sequence of an inventive process. An array of immobilized anti-probe spots 4 are provided on an array substrate 1. An anti-probe 4 is a nucleic acid having a sequence complementary to a single-strand oligonucleotide used herein as a nucleic acid probe 8. The anti-probe is readily immobilized on the substrate 1 through conventional techniques illustratively including covalent attachment to a functional group on the solid surface or biotinylation. It is appreciated that while an anti-probe 4 in simplest form includes only a single sequence complementary to a nucleic acid probe 8, an anti-probe having repeated sequences along the length thereof allows one to decrease anti-probe density within a spot, increase the number of nucleic acid probes 8 that can be hybridized to a spot, or a combination thereof. An anti-probe formed as a strand having multiple sequences complementary to nucleic acid probes is amenable to securement to a substrate 1 in an area to define a spot at one end or at both ends of the strand. After immobilizing anti-probes 4 onto substrate 1, the anti-probes are hybridized to complementary nucleic acid probes. Preferably, the anti-probes in a given spot 3 are known and vary in sequence relative to other spots on the array 1. Nucleic acid probes 8 complementary to nucleic acid anti-probe 4 are readily collected from a random mixture of short nucleic acid oligonucleotides or alternatively selected from a library. With the introduction of a solution of nucleic acid probes 8 into contact with array substrate 1 having immobilized anti-probes thereon under hybridization conditions, a double-stranded complex of anti-probe oligonucleotide with a complementary nucleic acid probe is formed in the multiple spots 3. It is appreciated that the anti-probe 4, in addition to being immobilized on a surface of array substrate 1, is readily isolated in a solution volume by porous media that is exclusive of the anti-probe in strand form while being permissive to nucleic acid probe movement. Alternatively, an anti-probe strand is isolated in a gel that is permissive to nucleic acid probe transport.
With the formation of double-strand complex of immobilized anti-probe 4 and free nucleic acid probe 8 to form spots 3 on array substrate 1, the array substrate 1 is placed in an array chamber 2. The array chamber 2 is in fluid communication with a target nucleic acid chamber 6 containing a target nucleic acid 5. In the lower left and right corners of each of FIGS. 1A-1F, cross-sectional, nonscaled images of the strandedness of the anti-probe 4 on substrate 1, and target nucleic acid 5, respectively, are provided.
The array chamber 2 and target chamber 6 are flooded with electrophoretic buffer solution and brought into fluid communication by way of a channel 7 through which nucleic acid probes 8 are amenable to transport while the target nucleic acid 5 is excluded from transport or at least travels through the channel 7 at a rate of less than 10% of the rate of nucleic acid probe movement. As used herein, a nucleic acid probe typically has a length of between 5 and 60 single-strand bases, and preferably between 5 and 50 bases. In contrast to the short nucleic acid probe oligonucleotides, a target nucleic acid 5 typically has a length of greater than 200 single-strand bases. It is appreciated that a target nucleic acid is present within target chamber 6 in a variety of forms. These forms include free-floating nucleic acid, as is conventional to the art; adhered to a surface of a substrate 9; attached to a particle such as a paramagnetic particle, a metal particle, semiconductor particle, or polymeric bead; or trapped within a volume by a size exclusive porous media permissible to nucleic acid probes; or trapped within a gel. As a result, it is appreciated that a DNA chip having target nucleic acid sequences adhered to a surface of a substrate 9 is operative with an inventive assay assemblage.
While the nature of the media within channel 7 can include size exclusive membranes, chromatographic media or gels, in a preferred embodiment the media within channel 7 is a gel amenable to electrophoresis. It is appreciated that various gels are now commonly used for a nucleic acid electrophoresis, these gels illustratively including polyacrylamide and agarose.
After forming the assay assemblage of FIG. 1A, electrophoretic electrodes 10 and 12 are introduced into the array chamber 2 and target chamber 6, respectively. The electrodes 10 and 12 are then coupled to an electrophoretic power supply 14. After denaturing the double-stranded anti-probe oligonucleotide-nucleic acid probe complexes arrayed in spots 3, the power supply 14 is energized to move the nucleic acid probes 8 through the channel 7. Demobilized anti-probes 4 remain adhered to substrate 1. Electrophoresis occurs until the nucleic acid probes 8 reach the target chamber 6 and the ability to interact with target nucleic acid 5.
Throughout FIG. 1 like numerals are used throughout to depict the movement and status of various components throughout the sequence of an inventive process.
The array of double-stranded complex spots 3 after denaturation and transport of nucleic acid probes 8 are now single-stranded anti-probe 4 remaining in position and denoted as white spots at 15 in FIG. 1B.
After establishing hybridization conditions within the target chamber to form a target nucleic acid-nucleic acid probe double-stranded complex, some nucleic acid probes 8 are hybridized to target nucleic acid 5 while other probes remain single stranded and in solution. Upon again establishing an electrophoretic potential between array chamber 2 and test chamber 6 with a reversed polarity relative to that depicted in FIG. 1B, those nucleic acid probes 8 which are not complementary in sequence to the target nucleic acid 5 remain single stranded in solution. These single-stranded free nucleic acid probes then migrate under the influence of the electric field to return to the assay chamber 2. After establishing hybridization conditions within the assay chamber 2, double-stranded anti-probe-nucleic acid probe complexes are formed only in those spots where the nucleic acid probe was not complementary for a sequence of the target nucleic acid 5. Subsequent to establishing hybridization conditions within assay chamber 2, a mixture of double-strand spots 3 and single-strand spots 15 exists on the substrate 1. The detection as to which nucleic acid probes derived from the array substrate 1 which are complexed to target nucleic acid 5 are identified by a number of methodologies conventional to the DNA chip art that involve fluorescent or other spectroscopic interrogation of target DNA. Preferably, an inventive assemblage according to FIG. 1 is developed to determine if the nucleic acid probe associated with a given spot is present on the substrate 1 by introducing a dye species that distinguishes between single-strand and double-strand compositions within each of the spots with knowledge as to the specific sequence of each anti-probe spotted on the array of substrate 1, as shown in FIG. 1E.
It is appreciated that the array of substrate 1 as depicted in FIG. 1A is regenerated to the original state after determination of homology between target nucleic acid and anti-probe according to FIG. 1E by denaturing the target nucleic acid double-stranded complex with complementary nucleic acid probes 8 and then repeating the electrophoretic migration of FIG. 1D and the nucleic acid probe-anti-probe hybridization of FIG. 1E followed by washing to remove single-stranded structure resolving dye present within the assay chamber 2.
An alternative determination of complementary nucleic acid probe identity to target nucleic acid is provided by the inclusion of a separate nucleic acid probe egress pathway. An inventive assemblage containing an egress pathway is particularly well suited for instances where the identity of anti-probe sequences is unknown, detection techniques other than through the inclusion of a dye is desired, subsequent chemistry is to be performed on the nucleic acid probes, or a combination thereof.
An inventive assemblage inclusive of an egress pathway is depicted in FIGS. 2A-2E where like numerals correspond to those detailed above with respect to FIG. 1. The egress pathway 18 depicted in FIGS. 2A-2E is provided as a blank form labyrinth. It is appreciated that numerous other pathways are operative in formation of an egress pathway. These forms illustratively include linear, arcuate, acute angular, and combinations thereof.
Referring now to FIG. 2A, an inventive assemblage 100 as detailed with respect to FIG. 1A is provided along with the inclusion of an egress pathway 18. The egress pathway 18 is filled with electrophoretic buffer solution and has a series of spots 20 of immobilized anti-probe 22 immobilized onto a surface 24 of the pathway 18. A cross-sectional schematic with elements not to scale is provided in the upper left portion of FIGS. 2A-2E depicting the strandedness of the immobilized anti-probes 22. The anti-probes 22, like anti-probes 4, are also readily in a solution well excluding anti-probe movement by a porous media, or embedding within a gel such that the porous media or gel are permeable to nucleic acid probes coming in contact therewith.
FIG. 2B depicts the denaturation of the double-stranded complex between anti-probe 4 and nucleic acid probe 8 and the migration of nucleic acid probes within an electric field as detailed above with respect to FIG. 1B.
After allowing nucleic acid probes to enter a target chamber 6 and interact with target nucleic acid 5 under hybridization conditions, nucleic acid probes 8 complementary to target nucleic acid 5 form a double-stranded target-probe complex as shown in FIG. 2C and uncomplementary, single-strand nucleic acid probes 8 are then moved into the egress pathway 18 under the influence of an electric potential established between electrode 10 positioned within the array chamber 2 and electrode 12 positioned at the terminus 26 of the pathway 18. The power supply 14 supplies a potential between electrodes 10 and 12. A single-stranded nucleic acid probe 8 traveling along pathway 18 hybridizes to a complementary anti-probe 22 to form a double-stranded pathway anti-probe-nucleic acid probe double-stranded complex 22-8 by converting a single-stranded spot 20 into a double-stranded spot denoted at 30.
The detection as to whether a given spot is a single-stranded anti-probe 20 or a double-stranded complex spot 30 again is amenable to conventional dye techniques such as the inclusion of a dye selective for either a single-strand or double-strand structure is spatially resolve the nature of each spot. Preferably, the resolution of spots as to single-strand spots 20 or double-stranded complex spots 30 involves time of flight from a spot to a detector 32. Since the distance between a given spot 20 or 30 and a detector 32 is known, the spacing between successive spots is known, and the molecular weight of a nucleic acid probe, time of flight detection as to the strandedness of a given spot is readily performed. In the embodiment depicted in FIG. 2D, multiple electrophoretic paths are created that encompass within that path a nucleic acid probe detector. The detector 32 functions based on spectrophotometrics or a change in electrical signal associated with a nucleic acid probe movement past a detector sensing a property such as conductivity or an electrophoretic voltage change necessary to maintain total power supplied across the electrodes. FIG. 2E shows the detector output for each of the detector lines 1-7 depicted in FIG. 2D in which a solid line denotes the absence of a nucleic acid probe while the dashed line denotes the detection of a nucleic acid probe that previously was part of complex 28. The eliciting was a function of time that corresponds to the series of five spots potentially feeding signal to each of lines 1-7. It is appreciated that any effluent from egress path 18 including that leaving terminus 26 depicted in FIG. 2C or from any one of lines 1-7 is readily returned to array chamber 2 to allow nucleic acid probes noncomplementary to the target nucleic acid to rehybridize to the anti-probes 4. Additionally, the complex between nucleic acid probes and target nucleic acid within chamber 6 is readily denatured from the complementary nucleic acid probes returned to array chamber 2 by reversing the polarity of the electrophoresis between electrodes 10 and 12 relative to FIG. 2B. Subsequent to rehybridization of the complementary nucleic acid probes to respective anti-probes 4, the assemblage is returned to the status depicted in FIG. 2A. Following completion of a test and the decision not to return nucleic acid probes to array chamber 2, the spent array substrate 1 is removed along with the test chamber 6. A new array 1′ and new nucleic acid target 6′ are placed in the respective chambers and a subsequent test then performed. This mode of operation is depicted schematically in FIG. 3.
In an alternative embodiment, subsequent to hybridization between the target nucleic acid 5 and complementary nucleic acid probes 8 to form a double-strand complex, the noncomplementary nucleic acid probes are returned via channel 7 to array chamber 2 and thereafter the double-stranded complex between complementary nucleic acid probes and target nucleic acid 5 are denatured with the complementary nucleic acid probes entering egress pathway 18 to produce an opposite spot pattern relative to that depicted in FIGS. 2C and 2D as well as the opposite line outputs of FIG. 2E. In this operational mode, effluent containing target nucleic acid complementary probes are also optionally recycled to the array chamber 2 such that following rehybridization the array substrate 1 is returned to an original state. Cycling of an array is appreciated to be of considerable value in high throughput automated testing. This operational scheme is depicted schematically in FIG. 4 with the recharging of the original array, removal of target chamber 6 and performing a new test with a new target chamber 6′ containing a different or potentially different target nucleic acid.
While there are numerous techniques known to the art for denaturing a double-strand nucleic acid complex, illustratively including heating, changes in pH, changes in ionic strength, and combinations thereof, an additional mode of inducing complex denaturation is detailed with respect to FIGS. 5A-5C. A neutral pH solution with both electrodes turned off represents a default state as shown in FIG. 5A, top panel. When both electrophoretic electrodes are activated, a sphere of low pH develops proximal to the electrodes as shown in FIG. 5A, lower panel. The electrolytic buffer solution of a high pH electrode activation creates a neutral pH region proximal to activated electrodes as shown in FIG. 5B, top panel. As a result, through adjustment of the buffer solution pH and switching electrodes between energized and deactive states allows a pH only in a vicinity of one electrode and double-stranded nucleic acid complex denaturation thereby releasing nucleic acid probe species from the spot in question, as shown in FIG. 5B, lower panel. Through the movement of a positive electrode downstream from a detector, free nucleic acid probe species migrate past a detector at a time indicative of the spot denoted in FIG. 5C as “electrode turned off.”
Top and cross-sectional views of a modular inventive assemblage well suited for manufacture to perform a process as depicted in FIGS. 1A-1E is shown generally at 60, where like numerals correspond to those used with respect to the preceding figures. The FIGS. 7A-7E are cross-sectional schematics of a process of operating the assemblage of FIGS. 6A and 6B, where lice numerals correspond to those used with respect to the preceding figures. In FIG. 7A, the manufacturer preloads of chambers with solutions. In FIG. 7B, a target nucleic acid sample is loaded into the target chamber. Thereafter, nucleic acid probes are electrophoretically transported and hybridized with target nucleic acid to which probe is complementary, as shown in FIG. 7C. Nucleic acid probes not complementary to the target nucleic acid are electrophoretically returned to the array chamber, as shown in FIG. 7D. Hybridization of those nucleic acid probes not complementary to the target nucleic acid to anti-probes immobilized and spotted in the array provides the identity of the probes sequences complementary to the target nucleic acid through imaging difference between single stranded anti-probes and double stranded probe-anti-probe complexes, as shown in FIG. 7E. The flow of washing and dye solutions through a conduit 62 allows one to determine strandedness in a given spot without opening the modular inventive assemblage 60.
In addition to the embodiments of the inventive assemblage depicted in FIGS. 1 and 2 in which a single array of probes bound to immobilized anti-probes interacts with a single test chamber and optionally includes an egress pathway, it is appreciated that the inventive concepts are readily extended to various combinations of probe arrays, target chambers, and detectors as depicted in FIGS. 8-11. While the embodiments depicted in FIGS. 9-11 include a detector at the rightmost extreme of the assemblage, it is appreciated that these embodiments as well as those depicted in FIG. 8 are readily modified to include one or more egress channels as detailed with respect to FIG. 2. Optionally, any number of lines as depicted in FIG. 2D are also provided to an egress pathway to facilitate alternate modes of detection.
Patent documents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These documents and publications are incorporated herein by reference to the same extent as if each individual document or publication was specifically and individually incorporated herein by reference.
The foregoing description is illustrative of particular embodiments of the invention, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the invention.
Patent Application
20080044821
