Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-13
2001-04-03
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S094000, C436S156000, C422S082010, C422S050000
Reexamination Certificate
active
06210896
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to molecular motors and their use in linear analysis of polymers. In particular, molecular motors are used to move polymers with respect to a station such that specific signals arise from the interaction between the polymer and an agent at the station.
BACKGROUND OF THE INVENTION
Polymers are involved in diverse and essential functions in living systems. The ability to decipher the function of polymers in these systems is integral to the understanding of the role that the polymer plays within a cell. Often the function of a polymer in a living system is determined by analyzing the structure and determining the relation between the structure and the function of the polymer. By determining the primary sequence in a polymer such as a nucleic acid it is possible to generate expression maps, to determine what proteins are expressed, and to understand where mutations occur in a disease state. Because of the wealth of knowledge that may be obtained from sequencing of polymers many methods have been developed to achieve more rapid and more accurate sequencing methods.
In general DNA sequencing is currently performed using one of two methods. The first and more popular method is the dideoxy chain termination method described by Sanger et al. (1977). This method involves the enzymatic synthesis of DNA molecules terminating in dideoxynucleotides. By using the four ddNTPs, a population of molecules terminating at each position of the target DNA can be synthesized. Subsequent analysis yields information on the length of the DNA molecules and the nucleotide at which each molecule terminates (either A, C, G, or T). With this information, the DNA sequence can be determined. The second method is Maxam and Gilbert sequencing (Maxam and Gilbert, 1977), which uses chemical degradation to generate a population of molecules degraded at certain positions of the target DNA. With knowledge of the cleavage specificities of the chemical reactions and the lengths of the fragments, the DNA sequence is generated. Both methods rely on polyacrylamide gel electrophoresis and photographic visualization of the radioactive DNA fragments. Each process takes about 1-3 days. The Sanger sequencing reactions can only generate 300-800 nucleotides in one run.
Methods to improve the output of sequence information using the Sanger method also have been proposed. These Sanger-based methods include multiplex sequencing, capillary gel electrophoresis, and automated gel electrophoresis. Recently, there has also been increasing interest in developing Sanger independent methods as well. Sanger independent methods use a completely different methodology to realize the nucleotide information. This category contains the most novel techniques, which include scanning electron microscopy (STM), mass spectrometry, enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA) sequencing, exonuclease sequencing, and sequencing by hybridization.
Further, several new methods have been described for carboxy terminal sequencing of polypeptides. See Inglis, A. S.,
Anal. Biochem.
195:183-96 (1991). Carboxy terminal sequencing methods mimic Edman degradation but involve sequential degradation from the opposite end of the polymer. See Inglis, A. S., Anal. Biochem. 195:183-96 (1991). Like Edman degradation, the carboxy-terminal sequencing methods involve chemically induced sequential removal and identification of the terminal amino acid residue.
More recently, polypeptide sequencing has been described by preparing a nested set (sequence defining set) of polymer fragments followed by mass analysis. See Chait, B. T. et al.,
Science
257:1885-94 (1992). Sequence is determined by comparing the relative mass difference between fragments with the known masses of the amino acid residues. Though formation of a nested (sequence defining) set of polymer fragments is a requirement of DNA sequencing, this method differs substantially from the conventional protein sequencing method consisting of sequential removal and identification of each residue. Although this method has potential in practice it has encountered several problems and has not been demonstrated to be an effective method.
SUMMARY OF THE INVENTION
The present invention relates to methods and products for linear analysis of polymers. In particular the invention is based on molecular motors and their use for guiding polymer movement during linear analysis. Recently rapid methods for analyzing polymers using linear analysis techniques have been developed. Such methods are described in co-pending PCT patent application No. PCT/US98/03024 and U.S. Ser. No. 09/134,411, the entire contents of which are hereby incorporated by reference. The method for analyzing polymers described in PCT/US98/03024 is based on the ability to examine each unit of a polymer individually. By examining each unit individually the type of unit and the position of the unit on the backbone of the polymer can be identified. This can be accomplished by positioning a unit at a station and examining a change which occurs when that unit is proximate to the station. The change can arise as a result of an interaction that occurs between the unit and the station or a partner and is specific for the particular unit. For instance if the polymer is a nucleic acid molecule and a T is positioned in proximity to a station a change which is specific for a T could occur. If on the other hand, a G is positioned in proximity to a station then a change which is specific for a G could occur. The specific change which occurs, for example, depends on the station used, the type of polymer being studied, and/or the label used. For instance the change may be an electromagnetic signal which arises as a result of the interaction.
One aspect of linear analysis techniques involves the movement of the polymer past a station in such a manner as to cause a signal that provides information about the polymer to arise. One method by which this movement can be achieved involves the use of molecular motors. A molecular motor is a molecule that interacts with a polymer and moves the polymer, unit by unit, past a station so that the polymer may be analyzed.
In one aspect the invention is a method for analyzing a polymer. The method includes the steps of exposing a plurality of individual units of a polymer to an agent selected from the group consisting of an electromagnetic radiation source, a quenching source, and a fluorescence excitation source by causing a molecular motor to move the polymer relative to the agent, and detecting signals resulting from an interaction between the units of the polymer and the agent. In one embodiment the signal is electromagnetic radiation. In another embodiment the agent is electromagnetic radiation.
In one embodiment the molecular motor is tethered to a support. Preferably the agent is also attached to the support. In another embodiment the agent is attached to the molecular motor.
In a preferred embodiment the agent is an electromagnetic radiation source. A portion of the plurality of individual units of the polymer, in one embodiment, is labeled with a fluorophore. In another embodiment the plurality of individual units of the polymer are sequentially exposed to electromagnetic radiation by bringing the plurality of individual units in proximity to a light emissive compound and exposing the light emissive compound to electromagnetic radiation, and wherein the plurality of individual units of the polymer detectably affect emission of electromagnetic radiation from the light emissive compound. Preferably the individual units detectably affecting emission of electromagnetic radiation from the light emissive compound are labeled with a fluorophore. According to another embodiment the plurality of individual units of the polymer are sequentially exposed to electromagnetic radiation, and wherein the electromagnetic radiation detectably affects emission of electromagnetic radiation from the plurality of individual units of the polymer to produce the detectable signal.
Th
Brusca John S.
Siu Stephen
US Genomics
Wolf Greenfield & Sacks P.C.
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