Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
1999-08-13
2001-07-17
Brusca, John S. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C435S006120
Reexamination Certificate
active
06263286
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to methods and apparatuses for analyzing molecules, particularly polymers, and molecular complexes with extended or rod-like conformations. In particular, the methods and apparatuses are used to identify repetitive information, e.g., sequence information, in molecules or molecular ensembles, which is subsequently used to determine structural information about the molecules. The methods are based on the use of an autocorrelation function to identify common information in multiple molecules having at least one overlapping repetitive sequence.
2. BACKGROUND OF THE INVENTION
Macromolecules are involved in diverse and essential functions in living systems. The ability to decipher the functions, dynamics, and interactions of macromolecules is dependent upon an understanding of their chemical and three-dimensional structures. These three aspects—chemical and three-dimensional structures and dynamics—are interrelated. For example, the chemical composition of a protein, and more particularly the linear arrangement of amino acids, explicitly determines the three-dimensional structure into which the polypeptide chain folds after biosynthesis (Kim & Baldwin (1990) Ann. Rev. Biochem. 59: 631-660), which in turn determines the interactions that the protein will have with other macromolecules, and the relative mobilities of domains that allow the protein to function properly.
Biological macromolecules are either polymers or complexes of polymers. Different types of macromolecules are composed of different types of monomers, i.e., twenty amino acids in the case of proteins and four major nucleobases in the case of nucleic acids. A wealth of information can be obtained from a determination of the linear, or primary, sequence of the monomers in a polymer chain. For example, by determining the primary sequence of a nucleic acid, it is possible to determine the primary sequences of proteins encoded by the nucleic acid, to generate expression maps for the determination of mRNA expression patterns, to determine protein expression patterns, and to understand how mutations in genes correspond to a disease state. Furthermore, the characteristic pattern of distribution of specific nucleobase sequences along a particular DNA polymer can be used to unequivocally identify the DNA, as in forensic analysis.
In general, DNA identification and sequencing has been performed using methods, such as those described by Maxam and Gilbert (Maxam & Gilbert (1977) Proc. Natl. Acad. Sci. U.S.A. 74: 560-564) and by Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467) that determine the exact sequence of relatively short pieces of DNA. There are also techniques that arrange these short DNA fragments of known sequence in the proper order to obtain a longer sequence, such as those described by Evans (U.S. Pat. No. 5,219,726). Other methods of nucleic acid detection and sequencing have been developed, however, these too have limitations in the number of nucleotides they can read, in their abilities to resolve the identities of adjacent nucleotides, and in the practicality of their implementation.
Several methods for rapid sequencing of nucleic acids have been developed that use exonucleases to cleave individual bases from the nucleic acid polymer, which are subsequently identified in order to generate the sequence of the nucleic acid. U.S. Pat. No. 4,962,037 discloses a method wherein the nucleic acid fragment is suspended in a flowing stream while an exonuclease sequentially cleaves individual bases from the end of the fragment. The flowing stream delivers the cleaved bases in an ordered fashion to a detector for subsequent identification. A similar approach with some modifications is disclosed in U.S. Pat. No. 5,674,743. In this method, the DNA strand to be sequenced is processed with an exonuclease to cleave bases from the strand, and each cleaved base is then transported away from the strand and is incorporated into a fluorescence-enchancing matrix. In a particular embodiment, the intrinsic fluorescence of the nucleotide is induced and is used to identify it. Using a processive exonuclease, it is theoretically possible to sequence 10,000 bases or more at a rate of 10 bases per second. However, exonuclease sequencing has encountered many problems. If extrinsic labels are used to identify each base, all four bases must be tagged with, e.g., different fluorophores, which is sterically difficult; in addition, introduction of fluorophores may interfere with the enzymatic activity of the exonuclease. Furthermore, difficult optical trapping is needed to suspend DNA molecules in a flowing stream. Lastly, single molecules of fluorophore need to be detected with high efficiency, and only 95% efficiency has been achieved.
Methods of nucleic acid sequencing by hybridization with a specific set of oligonucleotide probes are also known in the art (Strezoska et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88: 10089-10093; Bains (1992) BioTechnology 10: 757-758). Although this approach is very costly to set up, sequencing by these methods is ultimately low-cost ($0.03-0.08 per base). Another advantage is the potential integration of the technique with microelectronics using special microchips for sequencing of nucleic acids fragments and even analysis of entire genomes (Service (1998) Science 282: 396-399 & 399-401). Traditional sequencing by hybridization techniques have the limitation of imperfect hybridization, especially under conditions in which hybridization is not favored, e.g., low-salt, or upon formation of secondary structure in the target nucleic acid, which interferes with binding to the probes. Imperfect hybridization leads to difficulties in generating adequate sequence because the error in hybridization is amplified many times.
U.S. Pat. No. 5,846,727 discloses a microsystem for rapid DNA sequencing in which a DNA template is amplified using the polymerase chain reaction (“PCR”) and the PCR products are labeled and immobilized on a capillary tube wall. Then, Sanger extension products of the amplified DNA are prepared, labeled, and electrophoretically separated in a capillary channel. Near-infrared, laser-induced fluorescence of the oligonucleotides is detected. The same fluorophore is used to label all bases; however, different bases can be distinguished by difference of the fluorescence lifetimes induced by different bases upon the labeling. The substrate used is selected for compatibility with both the solutions and the conditions to be used in analysis, including but not limited to extremes of salt concentrations, acid or base concentration, temperature, electric fields, and transparence to wavelengths used for optical excitation or emission. The substrate material may include those associated with the semiconductor industry, such as fused silica, quartz, silicon, or gallium arsenide, or inert polymers such as polymethylmetacrylate, polydimethylsiloxane, polytetrafluoroethylene, polycarbonate, or polyvinylchloride. Because of its transmissive properties across a wide range of wavelengths, quartz is a preferred embodiment.
The use of quartz as a substrate with an aqueous solution means that the surface in contact with the solution has a positive charge. When working with charged molecules, especially under electrophoresis, it is desirable to have a neutral surface. In one embodiment, a coating is applied to the surface to eliminate the interactions which lead to the charge. The coating may be obtained commercially (capillary coatings by Supelco, Bellafonte Pa.), or it can be applied by the use of a silane with a functional group on one end. The silane end will bond effectively irreversibly with the glass, and the functional group can react further to make the desired coating. For DNA, a silane with polyethyleneoxide effectively prevents interaction between the polymer and the walls without further reaction, and a silane with an acrylamide group can participate in a polymerization reaction to create a polyacrylamide coating which not only does not
Chan Eugene Y.
Gilmanshin Rudolf
Brusca John S.
Lundgren Jeffrey S.
Pennie & Edmonds LLP
U.S. Genomics, Inc.
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