Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-03-09
2001-02-13
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S004000, C435S007800, C435S018000, C436S063000, C436S094000, C436S501000, C436S504000, C536S027400
Reexamination Certificate
active
06187566
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods of amplifying nucleic acids to minimize contamination by products of earlier amplification reactions. More particularly, it relates to methods of using nucleic acid labels that inhibit further amplification of the amplicon.
BACKGROUND OF THE INVENTION
Detecting specific gene sequences in clinical samples that are associated with disease states or biological conditions is frequently hindered by the low copy number of these gene sequences in the sample. The ability to replicate these gene sequences to improve sensitivity has revolutionized modem molecular genetics. There are currently many different methods for amplifying nucleic acids in samples to improve assay sensitivity, such as: polymerase chain reaction (PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202); ligase chain reaction (LCR); nucleic acid sequence-based amplification (NASBA) (U.S. Pat. Nos. 5,409,818 and 5,554,517); strand displacement amplification (SDA); and transcription-mediated amplification (TMA).
Many of these methods are capable of providing more than one billion copies of a single target nucleic acid in a very short time. Accordingly, one of the principle problems of using amplification technologies is that they are susceptible to contamination by exogenous nucleic acids. Although the latter can be controlled using careful laboratory techniques, the former source of contamination is hard to avoid in laboratories that repetitively amplify the same target sequences. In either case, this exogenous nucleic acid may be amplified along with the target nucleic acid in a clinical sample, which may lead to erroneous results.
Many different protocols have been developed in the past several years to prevent carryover contamination. Some of these protocols involve chemical, photochemical and/or enzymatic methods to inactivate the amplicons to prevent them from serving as templates in subsequent amplification reactions. When such methods are combined with appropriate laboratory techniques, the frequency of contamination-associated false-positive results is reduced. Since many of these types of decontamination protocols interfere with the amplification reaction, they must be carried out after amplification has been completed.
One of the more recently described methods for preventing contamination involves the use of UV irradiation to photochemically modify amplicon nucleotide bases. Such irradiation in the presence of certain isopsoralen derivatives forms cyclobutane adducts with pyrimidine bases, and the nucleic acids with these modified bases are no longer capable of serving as templates for subsequent PCR (G. D. Cimino et al., Nucleic Acid Research, 19(l):99-107 (1990)). However, this method has been described as only being useful when carried out after amplification has been completed, since these base reactions are non-specific and the reactants may interfere with the integrity of the target nucleic acid and other reaction components (R. Y. Walder et al., Nucleic Acids Research, 21(18):4339-4343 (1993)). Moreover, most of these currently used methods are adapted for use in PCR. Thus, it is not well established that such methods are equally as effective in other types of amplification reactions. Nor have decontamination protocols been specifically designed to be carried out during any stage of the amplification reaction.
Detection of the amplified nucleic acids involves the use of a labeling compound or compounds that can be measured and quantified. Many such labeling compounds are well known in the art. However, every additional step in the amplification reaction introduces additional reagent costs and assay time. Recently, methods have been develop that allow for simultaneous labeling and decontamination using reagents that are capable of serving both purposes.
Accordingly, there is a need to provide for improved decontamination reagents and protocols that are adapted for use before the amplification reaction has been completed, and that are suitable for simultaneous decontamination and labeling.
SUMMARY OF THE INVENTION
The present invention provides compositions that are useful for labeling and decontaminating a nucleic acid amplification reaction product, also referred to herein as an “amplicon”. Such compositions comprise “LACs”, which are covalent or noncovalent complexes of a binding ligand, a binding enhancer and a label. As described herein, the binding ligand is a chemical moiety that binds to the amplicon and that, when activated by light, forms at least one covalent bond therewith. Also as described herein, the binding enhancer is a chemical moiety that has a specific affinity for nucleic acids when compared to its affinity for the non-nucleic acid components of amplification reactions. As provided herein, the label is a detectable chemical moiety, such as a fluorophore, a chemiluminescent label or other chromophore.
In one aspect of the present invention, the binding ligand is either an intercalator compound, such as a furocoumarin or a phenanthridine, or a nonintercalator compound, such as a benzimide, a netropsin or a distamycin. When the binding ligand is an intercalator compound, in a preferred embodiment, it is an angelicin derivative.
In another aspect of the present invention, the binding enhancer is also either an intercalator compound or a nonintercalator compound, such as an oligo pyrrole, a phenyl indole, a nucleic acid or a protein.
In one embodiment, the LAC of the present invention is a complex of at least two intercalator moieties and a label.
Another aspect of the present invention is a method for labeling or decontaminating a nucleic acid amplification reaction product comprising the steps of preparing a nucleic acid amplification reaction mixture, contacting the mixture with the compositions just described, and exposing the mixture to light of an appropriate length of time and wavelength to cause the binding ligand to become covalently attached to the nucleic acid amplification reaction product.
Other aspects of the invention are described throughout the specification.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of amplifying a target analyte nucleic acid to produce multiple copies of the target, i.e. the nucleic acid reaction products of the amplification reaction which are also referred to as “amplicons”, and contacting the amplicons with a photoreactive compound, or “light-activated compound” (“LAC”) that serves the dual purpose of labeling and “deactivating” the amplicons. The present invention also relates to compositions comprising such LACs. By “deactivating”, it is meant that the photo-activated amplicons can no longer be amplified. In particular, the LAC is added to the amplification reaction before, during or after the nucleic acid amplification reaction. After the amplification reaction is completed, the reaction mixture is exposed to light of an appropriate wavelength to cause the labeling compound to become covalently linked to the amplicon. Thereafter, the amplicon is incapable of serving as a template for polymerization and thus prevented from contaminating subsequent amplification reactions.
Definitions
The following definitions are provided to further describe various aspects of the preferred embodiments of the present invention.
The term “amplification” is used to refer to a method for exponentially duplicating a target analyte nucleic acid in a sample to improve assay sensitivity. As described herein, many different methods for amplifying nucleic acids are known in the art. It should be understood that the particular amplification method employed in the practice of the present invention can vary depending on the type of target analyte, the type of sample, the desired sensitivity, and the like. The selection and performance of such amplification methods arc not within the scope of the present invention.
The term “binding ligand” is used to refer to a compound that has an affinity for nucleic acids, such that it forms a reversible complex with nucleic acids, and is capable of bei
Dattagupta Nanibhushan
Sridhar C. Nagaraja
Wu Whei-Kuo
Applied Gene Technologies, Inc.
Jones W. Gary
Morrison & Foester LLP
Taylor Janell E.
LandOfFree
Method of labeling a nucleic acid amplicon with simultaneous... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of labeling a nucleic acid amplicon with simultaneous..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of labeling a nucleic acid amplicon with simultaneous... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2578576