Chemistry: analytical and immunological testing – Optical result
Reexamination Certificate
2001-02-14
2004-07-13
Ludlow, Jan (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
C436S086000, C436S094000, C436S172000, C435S006120
Reexamination Certificate
active
06762059
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to methods and apparatuses for characterization of single polymers. In particular, the invention relates to methods and apparatuses for determination of the velocities of single elongated polymers. The invention also relates to methods for determination of the length and molecular mass of single polymers. The invention further relates to methods of determining the distance between landmarks on single polymers.
2. BACKGROUND OF THE INVENTION
Analysis of the structure and dynamics of single macromolecules in a fluid sample has attracted considerable interest due in part to the rapid development of methodologies for the manipulation and detection of single macromolecules. For example, recent developments in experimental techniques and available hardware have increased dramatically the sensitivity of detection so that optical detection can be made of single dye molecules in a sample. Single dye detection can be done in aqueous solution, at room temperature (see, e.g., Weiss, 1999, Science 283: 1676-1683), and in very small volumes to reduce background. Such single-molecule based analytical methods are especially useful in the analysis of biological macromolecules, such as nucleic acid molecules and proteins. Single-molecule analytical methods require small amounts of sample, thereby alleviating tedious efforts in generating large amounts of sample material. For example, single-molecule analytical methods may allow analysis of the structure of nucleic acid molecules without amplification, e.g., by polymerase-chain reaction (PCR). Single-molecule analytical methods also allow analysis of individual molecules, and are thus particularly useful in the identification of structural and/or dynamical features without the effect of averaging over a heterogeneous population.
A single-molecule electrophoresis (SME) method which combines single molecule detection and electrophoresis has been reported for the detection and identification of single molecules in solution (Castro and Shera, 1995, Anal. Chem. 67: 3181-3186). In SME, sizing of single molecules is accomplished through determination of electrophoretic velocities by measuring the time required for individual molecules to travel a fixed distance between two laser beams. This method has been applied to DNA, to fluorescent proteins and to simple organic fluorophores. For example, SME offers a single-molecule method for sizing of DNA restriction fragments. However, SME detects only the presence or absence of a molecule. The method does not provide information regarding the internal structure of a molecule.
A single-molecule DNA sizing method using a microfabricated device has also been reported (Chou et al., 1999, Proc. Natl. Acad. Sci. USA 96:11-13). The method makes use of the fact that the amount of intercalated dye is proportional to the length of the molecule, and determines the lengths of single DNA molecules by measuring the total fluorescence intensity of DNA stained with intercalating dye molecules. Thus, the method does not use electrophoretic mobilities to determine sizes of molecules. This method also does not provide information regarding the internal structure of a molecule.
PCT Publication No. WO 98/10097 discloses a method and apparatus for detection of single molecules emitting two-color fluorescence and determination of molecular weight and concentration of the molecules. The method involves labeling of individual molecules with at least two fluorescent probes of different emission spectrum. Simultaneous detection of the two labels indicates the presence of the molecule. The velocity of the molecule is determined by measuring the time required for the molecules to travel a fixed distance between two laser beams. Comparison of the molecule's velocity with that of standard species permits determination of the molecular weight of the molecule, which may be present in a concentration as small as one femtomolar.
Other techniques for characterizing single macromolecules include a method described in U.S. Pat. No. 5,807,677 for direct identification of a specific target nucleic acid sequence having a low copy number in a test solution. This method involves the preparation of a reference solution of a mixture of different short oligonucleotides. Each oligonucleotide includes a sequence complementary to a section of the target sequence and is labeled with one or more fluorescent dye molecules. The reference solution is incubated with the test solution under conditions favorable to hybridization of the short oligonucleotides with the nucleic acid target. The target sequence is identified in the solution by detection of the nucleic acid strands to which one or more of the labeled oligonucleotides are hybridized. To amplify the fluorescence signal, a “cocktail” of different oligonucleotides are used which are capable of hybridizing with sequences adjacent to but not overlapping with the target sequence. The disadvantage of this method is that, in order to design probes of the proper sequence, the exact sequence of the target nucleic acid and surrounding sequences must be known. A method described in U.S. Pat. No. 5,599,664 and European Patent No. EP 0391674 allows sizing of DNA molecules by first subjecting a DNA molecule to a force such that the DNA molecule is elongated and then measuring the conformational relaxation dynamics. In another method (Schmalzing et al., 1998, Analytical Chemistry 70:2303-2310; Schmalzing et al, 1997, Proc. Natl. Acad. Sci. USA 94:10273-10278), microfabricated devices for DNA analysis were developed, including sequencing, which employ small-scale versions of traditional techniques, such as electrophoresis.
None of these single molecule analytical methods allows the determination of the internal structure of the molecule. A challenge to the characterization of the internal structure, e.g., the linear sequence of monomers, in a single polymer chain is from the natural tendency of polymers in most media to adopt coiled conformations. The average degree of such coiling is dependent on, inter alia, the interaction of the polymer with the surrounding solution, the rigidity of the polymer, and the energy of interaction of the polymer with itself. In most cases, the coiling is quite significant. For example, a &lgr;-phage DNA, with a B-form contour length of about 16 &mgr;m long, has a random coil diameter of approximately 1 &mgr;m in water (Smith et al., 1989, Science 243:203-206).
Methods of elongating DNA molecules by fluid flow have been reported (Perkins et al. Science 276:2016-2021; Smith et al., Science 283:1724-1727). In one method, DNA molecules are stretched by an elongational flow. The probability distribution of molecular extension was determined as a function of time and strain rate. Detailed dynamics of elongated DNA molecules in elongational flow has also been observed. In another method DNA molecules are stretched by a steady shear flow. The probability distribution for the molecular extension was determined as a function of shear rate. It was found that, in contrast to the behavior in pure elongational flow, the average polymer extension in shear flow does not display a sharp coil-stretch transition.
DNA has also been stretched by electrophoresis as part of a near-field detection scheme for sequencing biomolecules. DNA has been elongated by electrophoresis both in a gel and in solution, using electrical forces to move the DNA in position for reading (U.S. Pat. No. 5,538,898). However, no data were given to determine the quality of the stretching of large polymers, and the technique is limited to analyzing approximately 3 megabases at a time.
Gravitational forces have also been used to stretch DNA (U.S. Pat. No. 5,707,797; Windle (1993) Nature Genetics 5:17-21). In this technique, drops of DNA from the sodium dodecyl sulfate lysing of cells were allowed to run down a slide held at an angle. The effect of gravity was enough to stretch out the DNA, even to its over-stretched S-DNA form. The DNA was then immobilized on the slide, making p
Chan Eugene Y.
Gleich Lance C.
Wellman Parris S.
Ludlow Jan
U.S. Genomics, Inc.
Wolf Greenfield & Sacks P.C.
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