Sample processing to release nucleic acids for direct detection

Details Sample processing to release nucleic acids for direct detection Sample processing to release nucleic acids for direct detection

1999-08-26

2001-06-05

Riley, Jezia (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C435S243000, C435S259000

Reexamination Certificate

active

06242188

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the field of nucleic acid detection and, more specifically, to the processing of samples to release nucleic acids in a condition suitable for direct detection.
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. Methods to detect nucleic acids commonly requires several sample processing steps, including use of a lysis reagent to lyse cells and release the nucleic acids contained within the cells. Lysis reagents typically consist of a strong detergent such as sodium dodecyl sulfate and alkaline pH conditions.
The need for multiple processing steps when using a lysis reagent, such as one containing a strong detergent, primarily results from inhibitors of later nucleic acid detection steps that are present or associated with the lysis reagent. The inhibitors must be neutralized or removed before amplification or other additional steps in nucleic acid detection can proceed. These additional steps result in increased labor and materials costs for the clinical laboratory. Use of a lysis reagent for nucleic acid detection also is detrimental because it can, under some circumstances, degrade the nucleic acids, thereby decreasing sensitivity in some assay formats. Thus, a need exists for an approach to isolate nucleic acids from a cell sample that avoids the additional steps associated with lysis reagents and allows for release and detection from a single reagent addition step.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to eliminate the additional processing steps and degradation associated with nucleic acid lysis procedures. This is achieved by using lipids that are non-denaturing for enzymes and proteins required in further processing steps.
It is also an object of the present invention to provide compositions for releasing nucleic acid from cells or samples that include reagents for labeling or performing amplification such that release and detection of nucleic acid can be performed by a single reagent addition step.
To accomplish these and other objectives, there has been provided, according to one aspect of the present invention, a composition comprising an aqueous solution for releasing nucleic acid from a sample for direct detection, comprising one or more lipids and, one or more of: i) an enzyme(s) to degrade cell structure; ii) a non-ionic membrane fluidizing compound(s); and iii) a metal chelator(s). The aqueous solution is non-inhibitory of enzymes or proteins that are used in nucleic acid release, amplification, labeling or detection, and can include one or more nucleic acid probes or primers complementary to the nucleic acid to be detected.
According to one embodiment of the present invention, the lipids of the aqueous solution comprise lipids in the form of liposomal vesicles or other structure for encapsulating the aqueous solution.
According to another embodiment of the present invention, the aqueous solution includes reagents for labeling nucleic acid. Such reagents comprise a compound comprising a photoactivatible binding ligand, a label comprising a detectable moiety and, optionally, a nucleic acid binding enhancer moiety.
According to yet another embodiment of the present invention, the aqueous solution further comprises one or more nucleic acid probes or primers complementary to the nucleic acid to be detected.
According to still yet another embodiment of the present invention, the one or more lipids of the aqueous solution comprise 3-(2-aminopropyl-1,3-dihexadecyloxypropyl) hexadecyl ether, 3-(2aminopropyl-1-octadecyloxy-3-benzyloxypropyl) benzyl sulfide, or bis(3-benzyloxypropyl-1-octadecyloxy-3-benzyloxy-2-propyl amine)-polyethyleneglycol.
In another aspect of the present invention, there is provided a composition comprising an aqueous solution comprising one or more membrane fluidizing compounds for releasing nucleic acid and one or more of: i) an enzyme(s) to degrade cell structure; ii) a lipid(s); and iii) a metal chelator(s). The aqueous solution is non-denaturing and non-inhibitory of enzymes or proteins that are used in nucleic acid release, amplification, labeling or detection.
According to one embodiment of the present invention, the lipids of the aqueous solution comprise lipids in the form of liposomal vesicles or other structure for encapsulating the aqueous solution.
According to another embodiment of the present invention, the aqueous solution includes reagents for labeling nucleic acid. Such reagents comprise a compound comprising a photoactivatible binding ligand, a label comprising a detectable moiety and, optionally, a nucleic acid binding enhancer moiety.
According to yet another embodiment of the present invention, the aqueous solution further comprises one or more nucleic acid probes or primers complementary to the nucleic acid to be detected.
In accordance with another another aspect of the present invention, methods are provided for detecting the presence of a nucleotide sequence in nucleic acid of a sample using the aqueous solutions comprising a lipid or membrane fluidizing compound containing compositions of the present invention. Such methods are applicable to clinical specimens and are useful for diagnosing a variety of diseases and conditions.
In accordance with still yet another aspect of the present invention, kits are provided for releasing nucleic acid from a sample in a form suitable for directly detecting the nucleic acid. The kit comprises a vial containing an aqueous solution comprising one or more lipids for releasing nucleic acid from the cells and further comprising one or more of an enzyme(s) to degrade cell structure, a non-ionic membrane fluidizing compound(s) and a metal chelator(s). The aqueous solution is non-denaturing and non-inhibitory of enzymes or proteins used in nucleic acid release, amplification, labeling or detection.
In one embodiment, the kit further comprises or more nucleic acid probes or primers complementary to the nucleic acid to be detected, wherein said probes or primers are contained in the vial with the aqueous solution or are contained in one or more separate vials.
In another embodiment, the kit includes a means to prepare liposomes with the reagents supplied with the kit. In another embodiment, the kit further includes reagents for labeling nucleic acid, wherein said reagents are contained in the vial with the aqueous solution or are contained in one or more separate vials.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel compositions and methods for processing cell samples under conditions such that nucleic acids in cells or otherwise inaccessible to detection are released in a form suitable for direct nucleic acid detection assays. The compositions of the invention are designed to release nucleic acid from cells under conditions that do not result in denaturation of enzymes or proteins used in nucleic acid release, amplification, labeling 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 fl

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