Stabilization of nucleic acid amplification cocktails

Details Stabilization of nucleic acid amplification cocktails Stabilization of nucleic acid amplification cocktails

1999-08-26

2002-04-30

Siew, Jeffrey (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091100, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06379930

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the field of nucleic acid detection and, more specifically, to the preparation of stabilized cocktails of reagents for nucleic acid amplification.
BACKGROUND OF THE INVENTION
Nucleic acid detection through modern molecular biological techniques has revolutionized diagnosis of infections, cancer, inborn genetic errors, HLA typing, and forensic and paternity testing. Diagnosis is accomplished through any of a variety of nucleic acid detecting methods, including, for example, the polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription mediated amplification (TMA) reaction, nucleic acid sequence based amplification (NASBA) reaction, and strand displacement amplification (SDA) reaction.
Reagents used in nucleic acid detection methods are typically prepared separately as individual stock solutions and are combined to produce the cocktail just prior to its use. For example, in PCR, a cocktail of reagents contains a DNA polymerase, appropriate nucleoside triphosphates, primer(s), and an amplification buffer. Typically, the cocktail of reagents cannot be stored at 4° C. for an extended period of time, but must be made fresh just before use to avoid undesirable reactions during storage between the individual reagents such as non-specific DNA polymerization of the nucleoside triphosphates in the absence of a target template.
The requirement to prepare and quality control separate stock solutions of each reagent used in amplification increases the costs of nucleic acid detection in the clinical lab. Also, the requirement to add several reagents to make the cocktail just before use increases the likelihood of error in the clinical lab. Thus, there is a need for a stabilized cocktail of nucleic acid detection reagents that is stable for extended times at 4° C.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to eliminate the requirement for separate preparation and quality control of each reagent used in a nucleic acid amplification reaction by providing a cocktail of the reagents in which undesirable reactions during storage between the reagents are avoided.
To accomplish this and other objectives, there has been provided, according to one aspect of the present invention, a composition comprising a cocktail of reagents for performing nucleic acid amplification that avoids undesirable reactions during storage between the individual reagents, thereby stabilizing the cocktail upon storage, comprising one or more of the reagents necessary to perform nucleic acid amplification and an inhibitory concentration of a reversible inhibitor(s) of the undesirable reaction.
According to one embodiment, the cocktail of reagents comprises one or more of a nucleic acid polymerase or ligase and one or more of a nucleoside triphosphate(s), nucleic acid primer(s) and an amplification buffer.
According to another embodiment, the cocktail of reagents comprises a lipid, which can be in the form liposomal vesicles wherein the cocktail of reagents is encapsulated within the liposomes.
According to yet another embodiment, the cocktail of reagents comprises all the reagents necessary to perform a nucleic acid amplification reaction.
According to another embodiment, the inhibitor of the undesirable reactions upon storage is a nucleic acid binding ligand. The binding ligand can be an intercalator compound, which can be monoadduct forming. The intercalator compound can be a furocoumarin such as 4′-aminomethyltrioxsalen (“AMT”) or angelicin, or a phenanthridine. The binding ligand also can be a non-intercalating compound such as benzimides, netropsins and distamycins.
According to another embodiment of the present invention, a method of nucleic acid amplification is provided using the composition comprising a stabilized cocktail of reagents.
According to yet another embodiment of the present invention, a method for preparing a stabilized cocktail of reagents which avoids undesirable reactions that occur between the reagents upon storage is provided. The method includes adding the inhibitor(s) of the undesirable reactions to the cocktail of reagents, wherein the inhibitor is added to the cocktail at a concentration that is inhibitory to the reaction but at a concentration which will be non-inhibitory when the cocktail is later diluted for its intended use. The method further includes adding a lipid for releasing nucleic acid from cells. In such cases, the lipid is used to produce liposomal vesicles and the stabilized cocktail of reagents and the inhibitor are encapsulated within the vesicles.
According to still yet another embodiment, the method for preparing a stabilized cocktail of reagents includes reagents suitable for performing polymerase chain reaction, ligase chain reaction, transcription based amplification reaction, nucleic acid sequence based amplification reaction and strand displacement amplification reaction.
According to another embodiment of the invention, the method of preparing a stabilized cocktail is for a transcription based or amplification reaction or a ligase chain reaction and said inhibitor(s) is phosphate ion.
According to yet another embodiment, the method for preparing a stabilized cocktail includes a binding ligand as the inhibitor. The binding ligand can be an intercalator compound, which can be monoadduct forming. The intercalator compound can be a furocoumarin such as AMT. The binding ligand also can be a non-intercalating compound.
According to still yet another aspect of the present invention, kits for performing nucleic acid amplification using the stabilized cocktail of reagents are provided.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel compositions and methods for preparing a cocktail of reagents that avoids undesirable reactions during storage between the reagents by addition of a reversible inhibitor of the reaction. Such undesirable reactions include, for example, formation of primer dimers, degradation of primers by exonuclease activity of the polymerase and non-specific polymerization of nucleoside triphosphates and/or primers.
The reagent cocktail is stable because of the presence of the inhibitor, thus allowing the cocktail to be stored for later use in amplification. Amplification is achieved when the cocktail is appropriately diluted with the target template such that the concentration of reaction inhibitor is below its effective level while the concentration of the other reagents are at an effective level. The use of stabilized cocktail of reagents eliminates the cost of preparation and quality control associated with preparing individual stock solutions of each reagent required for a particular nucleic acid extraction and/or detection.
General Definitions
Oligonucleotide: Low molecular weight deoxyribo-, ribo-, copolymers of deoxyribo- and ribonucleic acids of chain lengths between 3 and 150. Such oligonucleotides can have modified nucleotide residues such as —O-methoxy, phosphorothio-, methylphosphonates and others known in art.
Primers: Usually oligonucleotides which are used for extension reaction by a nucleic acid polymerase after a template primer hybrid is formed. Such primers can carry sequences specific for transcription by an RNA polymerase.
Nucleic Acid Probe: Nucleic acid with substantially complementary sequences to the target nucleic acids for detection or capture from a mixture. Such probes can be labeled for detection or immobilized onto a solid support to enrich the target by capture. A probe can be an single stranded or partially double stranded and can be an oligonucleotide or a larger nucleic acid.
Membrane fluidizing compound: A chemical substance that renders a cell membrane fluid or flexible to facilitate release of cellular material into solution or uptake of extracellular contents. Compounds that induce pinocytosis in addition to fluidizing the membrane also are included within the meaning of a membrane fluidizing compound as used herein. A membrane fluidizing compound can be a lipid or a non-lipid and can be ionic

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