Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-24
2001-07-24
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C436S094000, C536S025300
Reexamination Certificate
active
06265170
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
The invention relates to methods of sequencing or assaying nucleic acids, and more particularly to methods of accurately assaying triplex and duplex nucleic acid hybridization complexes.
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.
A more recently developed process in which an electric field is applied to a biological sample is disclosed in U.S. Pat. No. 5,824,477 to Stanley. The Stanley patent discloses a process for detecting the presence or absence of a predetermined nucleic acid sequence in a sample. The process comprises: (a) denaturing a sample double-stranded nucleic acid by means of a voltage applied to the sample in a solution by means of an electrode; (b) hybridizing the denatured nucleic acid with an oligonucleotide probe for the sequence; and (c) determining whether the hybridization has occurred. The Stanley patent discloses the application of an electric field to the sample to be assayed for the limited purpose of denaturing the target sequence.
A more well-known type of hybridization assay is based on the use of fluorescent marking agents. In their most basic form, fluorescent intensity-based assays have typically comprised contacting a target with a fluorophore-containing probe, removing any unbound probe from bound probe, and detecting fluorescence in the washed sample. Homogeneous assays improve upon such basic assays, in that the former do not require a washing step or the provision of a non-liquid phase support.
Some assays have employed intercalating fluorophores to detect nucleic acid hybridization, based on the ability of such fluorophores to bind between strands of nucleic acid in a hybridization complex.
For example, U.S. Pat. No. 5,824,557 to Burke et al. discloses a method and kit for detecting and quantitating nucleic acid molecules. A preferred embodiment relies on the intercalation of a dye into a double-stranded nucleic acid helix or single-stranded nucleic acid. The dye fluoresces after intercalation and the intensity is a direct measurement of the amount of nucleic acid present in the sample. While the method of Burke et al. is purported to be useful for measuring the amount of nucleic acid in a sample, the non-specific binding between intercalator and nucleic acid upon which the method is based renders the method impractical for detecting specific binding, particularly under conditions where non-target nucleic acid duplexes are present.
U.S. Pat. No. 5,814,447 to Ishiguro et al. discloses an assay which is purported to improve upon assays that rely on non-specific interaction between intercalating agents and nucleic acid duplexes, such as Burke et al. and an earlier assay described by Ishiguro et al. in Japanese Patent Public Disclosure No. 237000/1993. The earlier development comprised adding an intercalating fluorochrome having a tendency to exhibit increased intensity of fluorescence when intercalated to a sample solution before a specific region of a target nucleic acid was amplified by PCR, and measuring the intensity of fluorescence from the reaction solution at given time intervals to detect and quantitate the target nucleic acid before amplification. The '447 patent attempted to improve upon the earlier development by providing an assay having improved specificity, characterized in that the probe is a single-stranded oligonucleotide labeled with an intercalating fluorochrome which is to be intercalated into a complementary binding portion between a target nucleic acid and a single-stranded oligonucleotide probe.
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. patent application Ser. Nos. 08/807,901 and 08/870,370, respectively filed Feb. 27, 1997 and Jun. 6, 1997. The researchers indicated that the fluorescent intensity of a one base-pair mismatched duplex differed from that of a perfectly matched duplex. Thus, the applications 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.
Despite the foregoing developments, a need has continued to exist in the art for a simple, highly sensitive, effective and rapid method for analyzing interaction between nucleic acids and/or nucleic acid analogs.
All references cited herein are incorporated herein by reference in their entireties.
SUMMARY OF THE INVENTION
The invention provides a method for assaying sequence-specific hybridization, said method comprising:
providing a target comprising at least one nucleic acid sequence;
providing a probe comprising a nucleic acid or nucleic acid analog sequence;
adding said probe and said target to a hybridization medium to provide a test sample;
applying a first stimulus to said test sample to provide a first stimulated test sample;
detecting a first signal from said first stimulated test sample, wherein said first signal is correlated with a binding affinity between said probe and said target;
calibrating said first signal against a reference signal exhibited by a reference sample comprising at least one reference probe combined with said target, wherein relative to said target, each of said probe and said at least one reference probe is a different member selected from the group consisting of a perfect match, a one-base mismatch, a two-base mismatch, a three-base mismatch, a one-base deletion, a two-base deletion and a three-base deletion; and
determining from said calibrating a first determination of an extent of matching between said probe and said target;
applying a second stimulus to said first stimulated test sample to provide a second stimulated test sample; and
detecting a second signal from said second stimulated test sample, wherein said second signal is correlated with said binding affinity between said probe and said target;
determining from said detecting of said second signal a second determination of said extent of matching between said probe and said target; and
comparing said first determination and said second determination.
Also provided is another method for assaying hybridization, said method comprising:
providing a target comprising at least one nucleic acid sequence;
providing a probe comprising a nucleic acid or nucleic acid analog sequence;
adding said probe and said target to a hybridization medium to provide a test sample;
measuring a first signal of a first condition of said test sample to provide a primary determination regarding hybridization between said probe and said target, wherein said first signal is correlated with hybridization between
Daksis Jasmine I.
Erikson Glen H.
Picard Pierre
Caesar Rivise Bernstein Cohen & Pokotilow Ltd.
Chakrabarti Arun K.
Ingeneus Corporation
Jones W. Gary
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