Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-21
2001-11-13
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100
Reexamination Certificate
active
06316201
ABSTRACT:
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.
REFERENCES:
patent: 4390403 (1983-06-01), Batchelder
patent: 4711955 (1987-12-01), Ward et al.
patent: 4908112 (1990-03-01), Pace
patent: 5126021 (1992-06-01), Grossman
patent: 5126022 (1992-06-01), Soane et al.
patent: 5164055 (1992-11-01), Dubrow
patent: 5171534 (1992-12-01), Smith
patent: 5181999 (1993-01-01), Wiktorowicz
patent: 5187085 (1993-02-01), Lee
patent: 5188934 (1993-02-01), Menchen et al.
patent: 5264101 (1993-11-01), Demorest et al.
patent: 5290418 (1994-03-01), Menchen et al.
patent: 5366860 (1994-11-01), Bergot et al.
patent: 5374527 (1994-12-01), Grossman
patent: 5468365 (1995-11-01), Menchen et al.
patent: 5498392 (1996-03-01), Wilding et al.
patent: 5512131 (1996-04-01), Kumar et al.
patent: 5552028 (1996-09-01), Madabhushi et al.
patent: 5567292 (1996-10-01), Madabhushi et al.
patent: 5571410 (1996-11-01), Swedberg et al.
patent: 5585069 (1996-12-01), Zanzucchi et al.
patent: 5593838 (1997-0
Caliper Technologies Corp.
Houtteman Scott W.
Murphy Matthew B.
LandOfFree
Apparatus and methods for sequencing nucleic acids in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Apparatus and methods for sequencing nucleic acids in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Apparatus and methods for sequencing nucleic acids in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2592706