Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-01-24
2004-11-09
Benzion, Gary (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C436S164000, C436S172000, C536S024330
Reexamination Certificate
active
06815163
ABSTRACT:
INTRODUCTION
This invention was made in the course of research sponsored by the National Institutes of Health. The U.S. Government may have certain rights in this invention.
BACKGROUND OF THE INVENTION
Mutagenic lesions in DNA frequently result from structural modifications of the heterocyclic bases (exocyclic adducts, free radical-induced modifications) and/or from complete removal of the base (abasic sites). Further, cellular processing of lesion containing DNA can lead to mismatches, additions, deletions or base pair substitutions. Thus, by lesion, it is meant to include mismatches, additions and deletions. A wide range of lesion-induced thermodynamic effects have been observed. Typically, the free energy stabilizing the duplex is reduced significantly by inclusion of the lesion. Methods of determining lesion effects on duplex free energy are limited. Typically, the effect of a DNA modification on the energetics of duplex formation is measured by comparison of independently measured association constants for the modified and unmodified duplex or by comparing Tm values, which are commonly but erroneously believed to represent thermodynamic stability. Therefore, there is a need for a simple, reproducible and sensitive method for rapidly screening for duplex stability.
The assays of the invention have two novel features which, when combined, provide a powerful and rapid method to assess the consequences upon duplex formation of perturbations to localized and/or global chemical features of nucleic acids. The first unique aspect is using two simultaneously competing equilibria to identify differences in equilibrium constants, between formation of two different nucleic acid duplexes. Typically, the effect of a DNA modification on the energies of duplex formation have been measured by comparison of independently measured association constants for the modified DNA and the unmodified duplex requiring two separate experiments. A second novel feature of the present invention is that these assays require only one experiment.
Furthermore, there is a large technical barrier for direct measurement of single duplex association events. In conventional titration experiments, a solution of one strand is added to a solution of its complement with formation of a duplex monitored by any of a variety of methods, including spectroscopic and calorimetric methods. To extract useful information from a conventional titration, the experiment must be devised such that a significant fraction of free titrant will be present throughout the titration. Satisfaction of this condition leads to the familiar sinusoidal shape of the titration curve. To satisfy this condition, typically the product of the initial titrate concentration, c, and the association constant, K, is in the range 10<cK<1000. Due to the high association constant for nucleic acid duplexes, the component concentration must be below the association constant, the components are likely to be too dilute to be detected by standard spectroscopic means. Having nucleic acids compete for duplex formation, as in the present method, creates a second equilibrium, referred to herein as a “competition” for duplex formation, that is measurable at essentially any concentration range. Thus, the concentrations can be tailored to virtually any method of detection. Further, the competition is measured directly from a single experiment rather than having to compare the results of two independently measured experiments.
Another innovative aspect of this invention is the ability to discriminate between the two duplexes being formed. In one embodiment of the present invention fluorescence energy transfer is used to facilitate this discrimination. A common spectroscopic method for monitoring duplex formation relies on the hyperchromicity of duplex formation. However, the extinction coefficients of duplexes of similar length is not a very sensitive reporter of the small differences in DNA content that are of most interest, as in the case of oligonucleotide duplexes with damage to only a single base. Even a technique such as circular dichroism is not sufficiently sensitive and also suffers from difficulties in interpretation of spectral variation which can be due to factors other than duplex formation. FET provides a unique, extremely sensitive, and essentially binary means of discrimination because only a duplex with both the donor and acceptor dyes will have the spectroscopic signature of the energy transfer.
Competitive equilibrium assays of the present invention are more widely adaptable to a variety of nucleic acid systems than are assays that are based on changes in intrinsic spectral characteristics of the dyes. For example, the FET assay of the invention is dependent only on the presence of the two dyes and is limited only by the necessity of modifying DNA to bear the dyes and the fact that the distance between the dyes increases substantially when the initial FET duplex is disrupted. Any spectral changes that accompany the disruption of the FET duplex can be easily treated during data analysis and do not cause any significant complication for the FET assay.
Thus, the present invention has a number of significant advantages over prior art techniques for determining duplex stability.
SUMMARY OF THE INVENTION
An object of the present invention is to provide methods for screening for nucleic acid duplex stability by competitive equilibria. In these methods, a solution is first produced containing a known amount of an initial or reference nucleic acid duplex with a known stability. The initial duplex is comprised of a first nucleic acid strand having a sequence, wholly or in part, homologous to a target strand and a second nucleic acid strand having a sequence, wholly or in part, complementary to the target strand. A series of additions of target strand are then made by titrating the solution with a second solution comprising a known concentration of the target nucleic acid strand. This target nucleic acid strand competes with the first nucleic acid strand for binding to a nucleic acid strand of the initial nucleic acid duplex. After each addition or titration, the solution is subjected to conditions which disrupt some or all of the nucleic acid duplexes and triplexes in the solution; subjected to conditions which promote duplex or triplex formation, and then monitored for any changes in the amount of initial nucleic acid duplex formed as a function of the amount of target nucleic acid strand added. This method can be used for extracting enthalpy data by controlling temperature during duplex or triplex formation and monitoring changes as a function of temperature so that a family of titration curves can be made and used to extract enthalpy (&Dgr;H°) data.
Another object of the present invention is to provide a for detecting a single nucleotide polymorphisms. In this embodiment of the invention, the initial nucleic acid duplex comprises a first and second nucleic acid strand, wherein the first or second strand of the duplex is designed to identify a single nucleotide polymorphism in a single- or double-stranded target nucleic acid sequence. In this method, the amount of the initial nucleic acid duplex in a solution is first determined. A fixed excess amount of a target nucleic acid strand is then added to the solution. The solution is then subjected to conditions which disrupt some or all duplexes or triplexes in the solution followed by conditions which promote duplex or triplex formation. The amount of initial duplex formed after addition of the target strand is then measured. This measured amount, after addition of the target strand, is indicative of the target strand containing the single nucleotide polymorphism.
Another object of the present invention is to provide methods for determining the concentration of a target nucleic acid strand which comprises adding a known volume and concentration of an initial nucleic acid duplex with a known stability to a known volume of a solution containing a target strand. Alternatively, a known volume of a solution of t
Breslauer Kenneth J.
Gelfand Craig A.
Plum G. Eric
Benzion Gary
Licata & Tyrrell P.C.
Rutgers, the State University
Tung Joyce
LandOfFree
Methods and kits for screening nucleic acid duplex stability does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods and kits for screening nucleic acid duplex stability, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods and kits for screening nucleic acid duplex stability will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3351213