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Claims  |
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What is claimed:
1. A microfluidic apparatus for determining relative positions in a target nucleic acid sequence that are occupied by a first nucleotide, comprising:
a microscale separation channel having first and second ends for separating nucleic acid fragments by size;
a first source containing a nested set of first nucleotide termination fragments of the target nucleic acid in fluid communication with the separation channel, wherein the fragments terminate at each of the positions in the target occupied by the
first nucleotide;
a second source containing a nested set of second nucleotide termination fragments of the target nucleic acid, in fluid communication with the separation channel, wherein the fragments terminate at each of the positions occupied by the second
nucleotide;
a means for mixing a first concentration of the first nested set with a second concentration of the second nested set in a first mixture, the first concentration being detectably different from the second concentration;
a means for injecting a portion of the first mixture into the separation channel and transporting the portion through the separation channel; and
a means for detecting separate fragments of the first and second nested sets in the separation channel.
2. The microfluidic system of claim 1, wherein
the first and second sources are in fluid communication with the separation channel via a common injection channel; and
the means for mixing and the means for injecting comprises a controlled electrokinetic material transport system for concomitantly transporting the first and second concentrations into the injection channel to produce the first mixture, and for
injecting a portion of the first mixture from the injection channel into and along the separation channel.
3. The microfluidic system of claim 2, wherein the controlled electrokinetic material transport system comprises an electrical controller separately and operably coupleD to the first and second ends of the separation channel and to the first and
second sources, the electrical controller being capable of delivering a separate voltage to each of the first and second ends of the separation channel and to each of the first and second sources.
4. The microfluidic system of claim 1, wherein at least the separation channel comprises a separation matrix disposed therein.
5. The microfluidic system of claim 4, wherein the separation matrix comprises an acrylamide polymer solution.
6. The microfluidic system of claim 5, wherein the acrylamide polymer solution comprises a dimethylacrylamide polymer.
7. The microfluidic system of claim 1, wherein each of the fragments in the first and second nested sets comprise a detectable moiety, and wherein the means for detecting comprises a detection system arranged to detect the detectable moiety
within the separation channel.
8. The microfluidic system of claim 7, wherein the detectable moiety is an optically detectable moiety.
9. The microfluidic system of claim 8, wherein the optically detectable moiety is a fluorescent moiety and the detection system comprises:
a light source directed at the separation channel for exciting the fluorescent moiety; and
a photodetector for detecting fluorescence emitted from the fluorescent moiety.
10. The microfluidic system of claim 9, wherein the fluorescent moiety is selected from a fluorescein derivative and a rhodamine derivative.
11. The microfluidic system of claim 1, further comprising a body structure having a top portion, a bottom portion, and an interior portion, wherein the separation channel is defined by the interior portion, and wherein each of the first and
second sources comprises a port disposed through the top portion into the interior portion, each port being in fluid communication with the separation channel.
12. The microfluidic system of claim 11, wherein:
the bottom portion comprises a first substrate having a planar upper surface;
the top portion comprises a second substrate having a substantially planar lower surface;
the interior portion is defined by mating the upper surface with the lower surface; and
the separation channel comprises a groove fabricated into at least one of the upper or lower surfaces.
13. The microfluidic system of claim 12, wherein each of the first and second substrates comprises a silica-based substrate.
14. The microfluidic system of claim 13, wherein each of the first and second substrates comprises glass.
15. The microfluidic system of claim 12, wherein each of the first and second substrates comprise a polymeric substrate.
16. The microfluidic system of claim 15, wherein the polymeric substrate is selected from the group of polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene, polyvinylchloride (PVC), polydimethylsiloxane (PDMS) and polysulfone.
17. The microfluidic system of claim 1, further comprising:
a third source of a third nested set of fragments of the target nucleic acid sequence in fluid communication with the separation channel;
a fourth source of a fourth nested set of fragments of the target nucleic acid sequence in fluid communication with the separation channel; and
wherein the fragments in each of the first, second, third and fourth nested sets terminates in a first, second, third and fourth nucleotide, respectively, each of the first, second, third and fourth nucleotides being different from each other.
18. The microfluidic system of claim 17, wherein the fragments in the first nested set terminate in an adenosine or a nucleotide derivative thereof, the fragments in the second nested set terminate in a thymidine or a nucleotide derivative
thereof, the fragments of the third nested set terminate in cytosine or a nucleotide derivative thereof, and the fragments of the fourth nested set terminate in guanosine or a nucleotide derivative thereof.
19. A microfluidic system for determining relative positions in a target nucleic acid sequence that are occupied by a given nucleotide, comprising:
a body structure comprising a top portion, a bottom portion and an interior portion;
a microscale separation channel disposed in the interior portion, the separation channel having first and second ends;
an injection channel disposed in the interior portion and intersecting the separation channel at a first intersection;
first and second reservoirs disposed in the body structure, each of the first and second reservoirs being in fluid communication with the injection channel on a first side of the intersection;
wherein the first reservoir contains a first nested set of fragments of the target nucleic acid sequence each of the fragments in the first nested set terminating at a different position occupied by the given nucleotide, and the second reservoir
contains a second nested set of fragments of the target nucleic acid sequence, each of the fragments in the second nested set terminating in a nucleotide different from the given nucleotide;
an electrical controller operably coupled to each of the first and second reservoirs and the first and second ends, for concomitantly transporting a first concentration of the first nested set and a second concentration of the second nested set
through the intersection the first and second nested sets forming a first mixture, injecting a portion of the first mixture into the separation channel and transporting the portion along the separation channel.
20. A method of determining positions in a target nucleic acid sequence occupied by a first nucleotide, comprising:
providing a microfluidic device that comprises:
a separation channel for separating nucleic acid sequences by size;
a first source containing a nested set of first nucleotide termination fragments of the target nucleic acid in fluid communication with the separation channel, wherein the fragments terminate at each of the positions in the target occupied by the
first nucleotide;
a second source containing a nested set of second nucleotide termination fragments of the target nucleic acid, in fluid communication with the separation channel, wherein the fragments terminate at each of the positions occupied by the second
nucleotide;
mixing a first concentration of the first nested set . . . the first concentration being detectablY different from the second concentration;
transporting a portion of the first mixture through the separation channel to separate the fragment in each of the first and second nested sets:
detecting fragments separated in the transporting seep and distinguishing fragments from the first nested set from fragments from the second nested set based upon their relative concentration;
determining the positions within the target nucleic acid sequence occupied by the first nucleotide by comparing the size of fragments in the first nested set to the size of fragments in the second nested set.
21. The method of claim 20, wherein the mixing step comprises electrokinetically transporting the first concentration from the first source to an injection zone and concomitantly electrokinetically transporting the second concentration from the
second source to the injection zone to form the first mixture, wherein the injection zone is in fluid communication with the first and second sources and the separation channel.
22. The method of claim 20, wherein the transporting step comprises electrokinetically transporting a portion of the first mixture from the injection zone to the separation channel and along the separation channel.
23. The method of claim 20, wherein the detecting step comprises detecting a detectable moiety associated with the fragments of the first and second nested sets as the fragments are transported along the separation channel.
24. The method of claim 23, wherein the detectable moiety is a fluorescent moiety, and the detecting step comprises directing an activation light at the separation column and detecting an emitted fluorescence from the fluorescent moiety.
25. The method of claim 20, further comprising the step of repeating the steps of mixing, transporting, detecting and determining and wherein each of the fragments in the first nested set terminate in a second nucleotide, the second nucleotide
being different from the first nucleotide.
26. The method of claim 25, further comprising the step of repeating the steps of mixing, transporting, detecting and determining and wherein each of the fragments in the first nested set terminate in a third nucleotide, the third nucleotide
being different from the first and second nucleotides.
27. The method of claim 26, further comprising the step of repeating the steps of mixing, transporting, detecting and determining and wherein each of the fragments in the first nested set terminate in a fourth nucleotide, the fourth nucleotide
being different from the first, second and third nucleotides.
28. A method of determining positions in a target nucleic acid sequence occupied by a given nucleotide, comprising:
providing a microfluidic device, the device comprising:
at least a first separation channel having at least first and second ends;
at least first injection channel intersecting the separation channel at a first intersection;
at least first and second reservoirs in fluid communication with the injection channel;
placing a first nested set of fragments of the nucleic acid sequence in the first reservoir, each fragment in the nested set terminating at a different position occupied by the given nucleotide;
placing a second nested set of fragments of the target nucleic acid in the second reservoir, each fragment in the second nested set terminating in a nucleotide different from the given nucleotide;
concomitantly flowing a first concentration of the first nested set and a second concentration of the second nested set, into the injection channel to form a first mixture, the second concentration being determinably different from the first
concentration;
injecting a portion of the first mixture into the separation channel whereupon each of the fragments in each of the first and second nested sets are separated by size;
distinguishing the fragments of the first nested set from the fragments of the second nested set by their relative concentration; and
determining positions occupied by the given nucleotide based upon the size of the fragments in the first nested set relative to the size of the fragments in the second nested set. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
The basic foundation of life is built around the transmission of information, whether from cell to cell or from generation to generation. The transmission of this information is carried out by fundamental building blocks of biological organisms
including proteins, nucleic acids and the like. Accordingly, attempts to understand biological processes, variations in those processes and effectors of those processes, have naturally focused upon these building blocks to provide the information
necessary for that understanding.
In the field of nucleic acid analysis, there have been developed a number of methods and systems for determining the sequence of nucleotides in a given nucleic acid polymer. For example, in the 1970s, Maxam and Gilbert developed a method of
sequencing nucleic acid polymers by the selective chemical cleavage of the overall polymer. Maxam and Gilbert, Proc. Nat'l Acad. Sci., 74:560-564 (1977). Specifically, labeled nucleic acids were preferentially and partially cleaved after one of the
four nucleotides to create a nested set of fragments terminating in the particular nucleotide. Different conditions are applied to cleave after each of the four nucleotides creating corresponding nested sets. The fragments produced from the four
different treatments were then separated in four different lanes on a conventional polyacrylamide slab gel. Reading the bands on the gel in ascending order, one essentially reads off the sequence of the nucleic acid.
A reverse approach was presented by Sanger et al., Proc. Nat'l Acad. Sci., 74:5463-5467 (1977), where the four nested sets of fragments of the nucleic acid polymer were produced by transcription in the presence one of four chain terminating
dideoxynucleotide analogs. In particular, transcription of a nucleic acid template strand in the presence of the four deoxynucleoside triphosphates (dNTPs) and one dideoxynucleoside triphosphate analog (ddNTP) results in the production of a nested set
of fragments terminating in the one ddNTP. Specifically, during transcription, the occasional incorporation of the ddNTP into the sequence terminates the transcription process at that nucleotide. This process is repeated with each of the four different
ddNTP analogs.
While these methods have proven effective in determining sequence information, the use of slab gels and the reading processes are laborious and time consuming. Smith et al., U.S. Pat. No. 5,171,534, reports the use of four dideoxynucleotide
analogs in sequencing operations, wherein each different dideoxynucleotide is labeled with a spectrally distinguishable fluorescent moiety, in the method of Sanger, above. The four nested sets are produced using these dideoxynucleotides, whereupon each
set bears a spectrally resolvable label. All four sets are then sized in a single pass through a gel filled capillary, permitting the separation of the fragments based upon size. Fragments from each set are then distinguished of from one another by
virtue of filtering optics specific for the emission spectra of each resolvable label.
Again, while the use of differently labeled nested fragment sets provides advantages over previously used systems, sequencing by these methods still requires a substantial amount of labor, as well as substantial expense in purchasing the
necessary equipment, e.g. separations and detection equipment. Further, different fluorescent labels typically have different excitation spectra. As such, use of a single excitation light source in exciting and detecting all of four different labels,
e.g., in the method of Smith et al., results in less than optimal quantum yields for each of the labels used. Specifically, the excitation light source is typically not optimized for all of the fluorescent groups.
The present invention, on the other hand, provides a substantially low cost method and system for sequencing nucleic acids, which system is readily automatable and integratable with upstream or downstream processes.
SUMMARY OF THE INVENTION
The present invention generally provides microfluidic devices and systems, as well as methods of using such devices and systems in the determination of the nucleotide sequences in target nucleic acids.
In a first aspect, the present invention provides a microfluidic system for determining relative positions in a target nucleic acid sequence that are occupied by a given nucleotide. The system comprises a microscale separation channel having
first and second ends for separating nucleic acid fragments by size. The system also comprises a first source of a first nested set of fragments of the target nucleic acid sequence, in fluid communication with the separation channel, where each of the
fragments in the first nested set terminates at a different position occupied by the given nucleotide. Also included is a second source of a second nested set of fragments of the target nucleic acid sequence in fluid communication with the separation
channel, where each of the fragments in the second nested set terminates in a nucleotide different from the given nucleotide. The system also comprises a means for mixing a first concentration of the first nested set with a second concentration of the
second nested set in a first mixture, where the first concentration being determinably different from the second concentration. Further, the system comprises a means for injecting a portion of the first mixture into the separation channel and
transporting the portion through the separation channel to separate the fragments from the nested sets. Also included is a detector for detecting the separate fragments of the first and second nested sets in the separation channel. In preferred
aspects, the transport of materials, e.g., nested sets of fragments, through the various channels is carried out using a controlled electrokinetic material transport system.
In another preferred aspect, the sources of the nested sets of fragments are integrated into a single body structure with the microscale channels of the microfluidic device or system.
In a related aspect, the present invention provides a method of determining positions in a target nucleic acid sequence occupied by a first nucleotide. The method comprises providing a microfluidic device according to the present invention. A
first concentration of the first nested set from the first source is mixed with a second concentration of the second nested set from the second source to form a first mixture, wherein the first and second concentrations being distinguishably different
relative to each other. A portion of the first mixture is transported through the separation channel to separate the fragments in each of the first and second nested sets. The fragments separated in the transporting step are detected and the fragments
from the first nested set are distinguished from fragments from the second nested set based upon their relative concentration. The relative position within the target nucleic acid sequence occupied by the first nucleotide is then determined by comparing
the size of fragments in the first nested set relative to the size of fragments in the second nested set.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 schematically illustrates the body structure and assembly of a microfluidic device incorporating a layered body structure.
FIG. 2 schematically illustrates the channel and port/reservoir geometry for a microfluidic device that is useful in performing sequencing operations.
FIGS. 3A-D illustrate comparative plots of fluorescent signal vs. time for fragments separated in the separation portion of the microfluidic device of FIG. 2.
FIG. 4 schematically illustrates an overall microfluidic system for performing sequence analysis of target nucleic acids.
FIG. 5 illustrates one example of a process or software program for identifying nucleotides that occupy given positions within a target nucleic acid sequence.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is generally directed to microfluidic systems and methods of using such systems in the determination of the nucleotide sequence of target nucleic acid sequences (referred to herein as the "target"). In particular, the
present invention provides methods and systems for determining the relative positions within a target nucleic acid sequence that are occupied by a given nucleotide, e.g., A, T, G or C. As used herein, the phrase "target nucleic acid sequence" is used to
describe a nucleic acid polymer for which the sequence of nucleotide monomers is sought. The target nucleic acid may include natural or unnatural nucleic acid monomers, DNA, RNA or derivatives thereof. In general, the methods and systems of the present
invention employ selected mixtures of nested sets of fragments of the target nucleic acid that is to be sequenced. Separation of these fragments by size and comparison of their relative concentrations permits the elucidation of the relative positions of
specific nucleotides within the overall target sequence.
As noted, the above-described sequencing methods are carried out within microfluidic devices or systems. As used herein, the term "microscale" or "microfabricated" generally refers to structural elements or features of a device which have at
least one fabricated dimension in the range of from about 0.1 .mu.m to about 500 .mu.m. Thus, a device referred to as being microfabricated or microscale will include at least one structural element or feature having such a dimension. When used to
describe a fluidic element, such as a passage, chamber or conduit, the terms "microscale," "microfabricated" or "microfluidic" generally refer to one or more fluid passages, chambers or conduits which have at least one internal cross-sectional dimension,
e.g., depth, width, length, diameter, etc., that is less than 500 .mu.m, and typically between about 0.1 .mu.m and about 500 .mu.m. In the devices of the present invention, the microscale channels or chambers preferably have at least one cross-sectional
dimension between about 0.1 .mu.m and 200 .mu.m, more preferably between about 0.1 .mu.m and 100 .mu.m, and often between about 0.1 .mu.m and 20 .mu.m. Accordingly, the microfluidic devices or systems prepared in accordance with the present invention
typically include at least one microscale channel, usually at least two intersecting microscale channels, and often, three or more intersecting channels disposed within a single body structure. Channel intersections may exist in a number of formats,
including cross intersections, "T" intersections, or any number of other structures whereby at least two channels are in fluid communication.
The body structure of the microfluidic devices described herein typically comprises an aggregation of two or more separate layers which when appropriately mated or joined together, form the microfluidic device of the invention, e.g., containing
the channels and/or chambers described herein. Typically, the microfluidic devices described herein will comprise a top portion, a bottom portion, and an interior portion, wherein the interior portion substantially defines the channels and chambers of
the device.
FIG. 1 illustrates an example of a two-layer body structure 10, for a microfluidic device. In preferred aspects, the bottom portion of the device 12 comprises a solid substrate that is substantially planar in structure, and which has at least
one substantially flat upper surface 14. A variety of substrate materials may be employed as the bottom portion. Typically, because the devices are microfabricated, substrate materials will be selected based upon their compatibility with known
microfabrication techniques, e.g., photolithography, wet chemical etching, laser ablation, air abrasion techniques, injection molding, embossing, and other techniques. The substrate materials are also generally selected for their compatibility with the
full range of conditions to which the microfluidic devices may be exposed, including extremes of pH, temperature, salt concentration, and application of electric fields. Accordingly, in some preferred aspects, the substrate material may include
materials normally associated with the semiconductor industry in which such microfabrication techniques are regularly employed, including, e.g., silica based substrates, such as glass, quartz, silicon or polysilicon, as well as other substrate materials,
such as gallium arsenide and the like. In the case of semiconductive materials, e.g., silicon, it will often be desirable to provide an insulating coating or layer, e.g., silicon oxide, over the substrate material, and particularly in those applications
where electric fields are to be applied to the device or its contents.
In additional preferred aspects, the substrate materials will comprise polymeric materials, e.g., plastics, such as polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON.TM.), polyvinylchloride (PVC), polydimethylsiloxane
(PDMS), polysulfone, and the like. Such polymeric substrates are readily manufactured using available microfabrication techniques, as described above, or from microfabricated masters, using well known molding techniques, such as injection molding,
embossing or stamping, or by polymerizing the polymeric precursor material within the mold (See U.S. Pat. No. 5,512,131). Such polymeric substrate materials are preferred for their ease of manufacture, low cost and disposability, as well as their
general inertness to most extreme reaction conditions. Again, these polymeric materials may include treated surfaces, e.g., derivatized or coated surfaces, to enhance their utility in the microfluidic system, e.g., provide enhanced fluid direction,
e.g., as described in U.S. patent application Ser. No. 08/843,212, filed Apr. 14, 1997 (Attorney Docket No. 17646-002610), and which is incorporated herein by reference in its entirety for all purposes.
The channels and/or chambers of the microfluidic devices are typically fabricated into the upper surface of the bottom substrate or portion 12, or the lower surface 20 of upper substrate 18, or a combination thereof, as microscale grooves or
indentations 16, using the above described microfabrication techniques. The top portion or substrate 18 also comprises a first planar surface 20, and a second surface 22 opposite the first planar surface 20. In the microfluidic devices prepared in
accordance with the methods described herein, the top portion also includes a plurality of apertures, holes or ports 24 disposed therethrough, e.g., from the first planar surface 20 to the second surface 22 opposite the first planar surface.
The first planar surface 20 of t | | |
