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  High speed parallel molecular nucleic acid sequencingUSPTO Application #: 20060292583Title: High speed parallel molecular nucleic acid sequencing Abstract: A method and device is disclosed for high speed, automated sequencing of nucleic acid molecules. A nucleic acid molecule to be sequenced is exposed to a polymerase in the presence of nucleotides which are to be incorporated into a complementary nucleic acid strand. The polymerase carries a donor fluorophore, and each type of nucleotide (e.g. A, T/U, C and G) carries a distinguishable acceptor fluorophore characteristic of the particular type of nucleotide. As the polymerase incorporates individual nucleic acid molecules into a complementary strand, a laser continuously irradiates the donor fluorophore, at a wavelength that causes it to emit an emission signal (but the laser wavelength does not stimulate the acceptor fluorophore). In particular embodiments, no laser is needed if the donor fluorophore is a luminescent molecule or is stimulated by one. The emission signal from the polymerase is capable of stimulating any of the donor fluorophores (but not acceptor fluorophores), so that as a nucleotide is added by the polymerase, the acceptor fluorophore emits a signal associated with the type of nucleotide added to the complementary strand. The series of emission signals from the acceptor fluorophores is detected, and correlated with a sequence of nucleotides that correspond to the sequence of emission signals. (end of abstract) Agent: Klarquist Sparkman, LLP - Portland, OR, US Inventors: Thomas D. Schneider, Denise Rubens USPTO Applicaton #: 20060292583 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060292583. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD [0001] This disclosure relates to an automated method for sequencing nucleic acids, such as DNA and RNA, which may be used for research and the diagnosis of disease in clinical applications. BACKGROUND [0002] Approaches to DNA sequencing over the past twenty years have varied widely. The use of enzymes and chemicals is making it possible to sequence the human genome. However, this effort takes enormous resources. [0003] Until recently, there were only two general sequencing methods available, the Maxam-Gilbert chemical degradation method (Maxam and Gilbert, 1977, Proc. Natl. Acad. Sci., USA 74:560), and the Sanger dideoxy chain termination method (Sanger et at, 1977, Proc. Natl. Acad Sci., USA 74:5463). Using the dideoxy chain termination DNA sequencing method, DNA molecules of differing lengths are generated by enzymatic extension of a synthetic primer, using DNA polymerase and a mixture of deoxy- and dideoxy-nucleoside triphosphates. To perform this reaction, the DNA template is incubated with a mixture containing all four deoxynucleoside 5'-triphosphates (dNNs), one or more of which is labeled with .sup.32P, and a 2',3'-dideoxynucleoside triphosphate analog (ddNTP). Four separate incubation mixtures are prepared, each containing a different ddNTP analog (ddATP, ddCTP, ddGTP, or ddTTP). The dideoxynucleotide analog is incorporated normally into the growing complementary DNA strand by the DNA polymese, through their 5' triphosphate groups. [0004] However, because of the absence of a 3'-OH group on the ddNTP, phosphodiester bonds cannot be formed with the next incoming dNTPs. This results in termination of the growing complementary DNA chain. Therefore, at the end of the incubation period, each reaction mixture contains a population of DNA molecules having a common 5' terminus, but varying in length to a nucleotide base specific 3' terminus. These four preparations, with heterogeneous fragments each ending in either cytosine (C), guanine (G), adenine (A) or thymine MT) are separated in four parallel lanes on polyacrylamide gels. The sequence is determined after autoradiography, by determining the terminal nucleotide base at each incremental cleavage in the molecular weight of the electrophoresed fragments. [0005] The Maxam-Gilbert method of DNA sequencing involves the chemical-specific cleavage of DNA. In this method, radio-labeled DNA molecules are incubated in four separate reaction mixtures, each of which partially cleaves the DNA at one or two nucleotides of a specific identity (G, A+G, C or C+T). The resulting DNA fragments are separated by polyacrylamide gel electrophoresis, with each of the four reactions fractionated in a separate lane of the gel. The DNA sequence is determined after autoradiography, again by observing the macromolecular separation of the fragments in the four lanes of the gel. [0006] The use of fluorescent nucleotides has eliminated the need for radioactive nucleotides, and provided a means to automate DNA sequencing. As fluorescent DNA fragments on an electrophoresis gel pass by a detector, the sequential fluorescent signals (which correspond to a fragment ending in a particular nucleotide) are automatically converted into the DNA sequence, eliminating the additional step of exposing the gel to film. Improvements on this general concept have been the subject of several U.S. patents, including U.S. Pat. No. 5,124,247 to Ansorge, U.S. Pat. No. 5,242,796 to Prober et al., U.S. Pat. No. 5,306,618 to Prober et al., U.S. Pat. No. 5,360,523 to Middendorf et al., U.S. Pat. No. 5,556,790 to Pettit, and U.S. Pat. No. 5,821,058 to Smith et al. However, the methods disclosed in these patents still require the inconvenient step of separating the generated DNA fragments by size, using electrophoresis. [0007] There are several disadvantages associated with using electrophoresis for nucleic acid sequencing. Electrophoresis requires macroscopic separation, with the necessity of expensive reagents, long gel preparation time, tedious sample loading, the dangers of exposure to the neurotoxin acrylamide. Macromolecular electrophoretic separation also exposes the technician to high voltage devices, requires prolonged electrophoresis time, produces gel artifacts, and requires calculations to adjust for dye mobilities. Furthermore, sequencing runs only allow for the sequencing of less than 1000 bases at a time, which can be a substantial drawback to the sequencing of long stretches of the genome. [0008] Given the practical drawbacks of electrophoresis, attempts have been made to eliminate this step. Mills, for example, described the use of mass spectrometry to separate the DNA fragments as an alternative to electrophoresis (U.S. Pat. Nos. 5,221,518 and 5,064,754). However, mass spectrometry devices are expensive, and because the method depends on size separation, it has a size resolution limit. [0009] Others have attempted to separate nucleic acid sequences by size using capillary electrophoresis (Karger, Nucl. Acids Res. 19:4955-62, 1991). In this method, fused silica capillaries filled with polyacrylamide gel are used as an alternative to slab gel electrophoresis. However, this method is limited by the separation process and requires very high detection sensitivity and wavelength selectivity due to the small sample size. [0010] Melamede (U.S. Pat. No. 4,863,849) and Cheeseman (U.S. Pat. No. 5,302,509) describe DNA sequencing methods which require a complex external liquid pumping system to add and remove necessary reagents. In these "open" systems, which contain the polymerase and the DNA to be sequenced, fluorescent nucleotides are pumped into a reaction chamber and added to the DNA molecule. After the incorporation of a single nucleotide, unincorporated fluorescent dNTPs are removed, leaving behind the DNA and its newly incorporated fluorescent nucleotide. This incorporated nucleotide is detected, its signal converted into a DNA sequence, and the process is repeated until the sequencing is complete. Although these methods can eliminate the electrophoresis step, the addition of nucleotides must be monitored one at a time as they are added to a population of DNA molecules, by continually pumping materials in and out of the reaction chamber. [0011] In another automated process, Jett e al. (U.S. Pat. Nos. 4,962,037 and 5,405,747) uses an exonuclease to sequentially shorten a DNA molecule that is being sequenced. After a complementary DNA strand is synthesized in the presence of fluorescent nucleotides, the exonuclease cleaves individual fluorescent nucleotides from the end of the synthesized DNA molecule. These nucleotides pass through a detector, and the fluorescent signal emitted by each nucleotide is recorded to determine the DNA sequence. [0012] In the methods of Melamede (U.S. Pat. No. 4,863,849) and Cheeseman (U.S. Pat. No. 5,302,509) described above, the addition or release of nucleotides from several DNA molecules is monitored simultaneously. This is sequencing at the macromolecular level, as opposed to sequencing at the molecular level, which involves monitoring the addition or release of nucleotides from a single DNA molecule. A disadvantage of macromolecular sequencing methods is that even though all of the DNA molecules start with identical nucleotides, they may quickly evolve into a mixed population. When using the macromolecular methods, some chains may more efficiently incorporate nucleotides than others, and some DNA may be degraded more slowly or rapidly than others. [0013] To solve this synchronization problem, Jett et al. (U.S. Pat. No. 4,962,037) and Ulmer (U.S. Pat. No. 5,674,743) developed molecular level sequencing systems in which a single fluorescently labeled DNA base is sequentially cleaved from a DNA molecule. The fluorescent signal from each cleaved dNTP is used to determine the DNA sequence. One drawback to these methods, however, is that the DNA molecule which is being sequenced must be held in a stream, which often results in shearing of the DNA, especially at higher flow rates. The sheared DNA molecule can not be accurately sequenced. In addition, only one DNA molecule can be sequenced at a time by this method. [0014] The development of fluorescence resonance energy transfer (FRET) labels for DNA sequencing has been described by Ju (U.S. Pat. No. 5,814,454) and Mathies et al. (U.S. Pat. No. 5,707,804). During FRET, exciting the donor dye with light of a first wavelength releases light of a second wavelength, which in turn excites the acceptor dye(s) to emit light of a third wavelength, which is then detected. These patents disclose the attachment of FRET labels to oligonucleotide primers for sequencing DNA molecules. A drawback of these methods is that there is still a need for size separation (for example using electrophoresis) prior to determining the DNA sequence. [0015] Therefore, there remains a need for a method of sequencing nucleic acids at the molecular scale, that does not require the use of electrophoresis or complex liquid pumping systems, and does not result in the shearing of nucleic acids. In addition, methods that are automated would be particularly useful. SUMMARY OF THE DISCLOSURE [0016] The present disclosure provides an improved method and device for sequencing nucleic acids. The method allows several nucleic acids to be sequenced simultaneously at the molecular level. In particular examples, the method uses a donor and acceptor class of dyes. This method and device minimize shearing the sample nucleic acids to be sequenced, and can be readily automated. [0017] Herein disclosed is a method of sequencing a sample nucleic acid molecule by exposing the sample nucleic acid molecule to an oligonuclootide primer and a polymerase in the presence of a mixture of nucleotides. The polymerase carries a fluorophore, and each different type of nucleotide (e.g. A, T/U, C or G) carries a fluorophore which emits a signal that is distinguishable from a signal emitted by the fluorophore carried by each of the other types of nucleotides. In particular embodiments the fluorophore on the polymerase is a donor fluorophore and the fluorophore carried on the nucleotides are acceptor fluorophores. The donor fluorophore can be excited by a source of electromagnetic radiation (such as a laser) that specifically excites the donor fluorophore and not the acceptor fluorophores. This excitation induces the donor to emit light at a wavelength that can transfer energy to excite only the acceptor fluorophores that are added to the complementary strand by the polymerase. As the donor fluorophore excites the acceptor, a signal characteristic of the specific nucleotide being added (e.g. A, T/U, C or G) is emitted by the acceptor fluorophore. A series of sequential signals emitted by the added nucleotides is detected, and converted into the complement of the nucleic acid sample. In particular embodiments, the unique emission signal for each nucleotide is generated by luminescence resonance energy transfer (LRET) or fluorescent resonance energy transfer (FRET). [0018] In other embodiments, the nucleic acid is a DNA or RNA molecule, and correspondingly, the polymerase is a DNA or RNA polymerase, if DNA is being sequenced, or reverse transcriptase if RNA is being sequenced. In a further embodiment, the polymerase is a Klenow fragment of DNA polymerase I. In particular embodiments, the polymerase is a GFP-polymerase. In another embodiment, the donor fluorophore is green fluorescent protein (GFP). In particular embodiments, the donor fluorophore, such as GFP, is excited by a laser. In other embodiments, GFP can be excited by a luminescent molecule, for example aequorin. [0019] Alternatively, the donor fluorophore is a luminescent molecule, for example aequorin or europium chelates. In this embodiment, the donor fluorophore does not require excitation by a source of electromagnetic radiation, because the luminescent donor fluorophore is naturally in an excited state. [0020] In yet another embodiment, the acceptor fluorophores are BODIPY, fluorescein, rhodamine green, and Oregon green or derivatives thereof. In particular, the donor fluorophore and one of the acceptor fluorophores comprise a donor/acceptor fluorophore pair selected from the group consisting of the GFP mutant H9-40, tetramethylrhodamine, Lissamine.TM., Texas Red and naphthofluorescein. [0021] Also disclosed herein are embodiments in which the polymerase may be fixed to a substrate, for example by a linker molecule that includes a polymerase component and a substrate component. The linker may be selected from the group consisting of streptavidin-biotin, histidine-Ni, S-tag-S-protein, and glutathione-glutathione-S-transferase (GST). In another embodiment, a nucleic acid may be fixed to a substrate. In particular embodiments the oligonucleotide primer is fixed to a substrate, for example at its 5' end. In yet other embodiments, the sample nucleic acid to be sequenced is fixed to the substrate. In particular embodiments, the sample nucleic acid to be sequenced is fixed to the substrate by its 5' end, 3' end or anywhere in between. In another embodiment, a plurality of polymerases, oligonucleotide primers, or sample nucleic acids arc fixed directly or indirectly to the substrate in a predetermined pattern. For example, the polymerases can be deposited into channels which have been etched in an orderly array or by micropipetting droplets containing the polymerases onto a slide, for example either by manually pipetting or with an automated arrayer. In other embodiments, a plurality of sequencing reactions are performed substantially simultaneously, and the signals from the plurality of sequencing reactions detected. Continue reading... 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