Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-29
2003-09-02
Benzion, Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S287200, C536S025300, C436S094000
Reexamination Certificate
active
06613524
ABSTRACT:
SPECIFICATION
BACKGROUND OF THE INVENTION
1. Field of Invention
The invention relates to the conductivity of nucleobase-containing sequences, such as DNA, RNA and analogues thereof, and more particularly to methods wherein the conductivities of such sequences are analyzed to reveal information about additional properties of the sequences.
2. Description of Related Art
It has been understood for a number of years that biological molecules can be isolated and characterized through the application of an electric field to a sample.
Electrophoresis is perhaps the most well-known example of an isolation and characterization technique based on the influence of electric fields on biological molecules. In gel electrophoresis, a uniform matrix or gel is formed of, for example, polyacrylamide, to which an electric field is applied. Mixtures applied to one end of the gel will migrate through the gel according to their size and interaction with the electric field. Mobility is dependent upon the unique characteristics of the substance such as conformation, size and charge. Mobilities can be influenced by altering pore sizes of the gel, such as by formation of a concentration or pH gradient, or by altering the composition of the buffer (pH, SDS, DOC, glycine, salt). One- and two-dimensional gel electrophoresis are fairly routine procedures in most research laboratories. Target substances can be purified by passage through and/or physical extraction from the gel.
In the ongoing search for more sensitive, accurate and rapid assay techniques, one research group developed an assay comprising analyzing the effects of an electric field on the fluorescent intensity of nucleic acid hybridization duplexes. See U.S. Pat. Nos. 5,846,729 to Wu et al., 6,060,242 to Nie et al. and 6,294,333 to Picard et al. The researchers indicated that the fluorescent intensity of a one base-pair mismatched duplex differed from that of a perfectly matched duplex or triplex when subjected to an electrical field. Thus, the patents purport to disclose a method for detecting a nucleotide sequence, wherein an electric field is applied to a liquid medium prior to or concurrently with a detecting step, and a change in an intensity of a fluorescent emission as a function of the electric field is detected as an indication of whether the probe is hybridized to a completely complementary nucleotide sequence or an incompletely complementary nucleotide sequence.
Others have studied the electrical properties of DNA more directly. For example, Porath et al., “Direct Measurement of Electrical Transport through DNA Molecules,” 403 Nature 635 (Feb. 10, 2000), discloses the current-voltage curves measured at room temperature on a DNA molecule trapped between two metal nanoelectrodes. Current was essentially zero up to a threshold voltage of about 1-2 volts. There is no suggestion in this reference that DNA properties other than electric transport can be correlated with current flow through DNA.
U.S. Pat. No. 5,783,063 to Clarkson et al. discloses a method for estimating a property of a nucleic acid material, which property is one to which the electrical conductivity of the nucleic acid material is related. The only properties detected are the concentration of the nucleic acid material in solution and the molecular weight of the nucleic acid material. The method comprises measuring the electrical conductivity of a solution containing the nucleic acid material and estimating from the measurement the concentration or molecular weight of the nucleic acid material by reference to calibration curves.
All references cited herein are incorporated herein by reference in their entireties.
BRIEF SUMMARY OF THE INVENTION
The invention provides a method for determining an affinity of a first nucleobase-containing sequence for a second nucleobase-containing sequence, the method comprising:
providing a test medium containing the first nucleobase-containing sequence and the second nucleobase-containing sequence, wherein the first nucleobase-containing sequence and the second nucleobase-containing sequence are of different lengths;
applying a voltage across the test medium;
measuring a test electric current through the test medium; and
determining the affinity by evaluating whether the test electric current is equivalent to a reference electric current of a reference medium containing a longer of the first nucleobase-containing sequence and the second nucleobase-containing sequence.
Also provided is a complex in an electrically-stimulated phase containing at least two nucleobase-containing sequences in a medium, wherein the electrical conductivity of the medium increases linearly without a plateau as the temperature of the medium approaches and exceeds a Tm of the complex.
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Benzion Gary
Caesar Rivise Bernstein Cohen & Pokotilow Ltd.
Chakrabarti Arun Kr.
Ingeneus Corporation
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