Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1994-11-30
2003-08-26
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S183000, C435S184000, C435S195000, C435S199000, C435S219000, C435S810000
Reexamination Certificate
active
06610475
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to procedures for treating biological specimens to make their nucleic acids available for various purposes, such as nucleic acid hybridization assays for the diagnosis of disease and other purposes, and for amplification of nucleic acids by the polymerase chain reaction (PCR) or other target amplification procedures. Specifically, the present invention relates to convenient procedures for making nucleic acids available that prevent degradation of the nucleic acids by endogenous nucleases present in the biological sample.
BACKGROUND OF THE INVENTION
Many diagnostic procedures are based on detection of specific nucleic acid (DNA or RNA) sequences present in a biological sample. For example, the sample may contain bacteria, viruses, or other microorganisms whose presence must be ascertained to determine the cause of an infectious disease. In other instances, the nucleic acid sequence may be sought within the DNA of a human white blood cell in order to establish the presence of a mutation associated with cancer or a genetic disease.
For such a diagnostic analyses, it is necessary to make available the specific nucleic acid that may be present in the sample. Frequently, the nucleic acid will be contained within a bacterium, fungus, virus, or other microorganism or within human cells such as white blood cells. It may further be contained within other structures such as ribosomes, plasmids, or chromosomal DNA. In order to perform hybridization reactions to detect specific nucleic acids or to amplify them using PCR or other target amplification methods, the nucleic acid must be released from these organisms and/or structures.
Unfortunately, such release exposes the nucleic acids to degradation by endogenous nucleases present in the sample, which may exist in such abundance that the nucleic acid is almost instantaneously destroyed.
The problem is particularly acute when the specific nucleic acid is an RNA, since RNAses are abundant in most biological samples and are often extremely resistant to treatments that readily inactivate many other enzymes.
To deal with this problem, it is common in the art to employ a variety of means to purify the nucleic acids from the biological sample. For example, anionic detergents and chaotropic agents such as guanidinium salts have been used to simultaneously inactivate or inhibit nuclease activities and release nucleic acids from within cells and subcellular structures. Unfortunately, these agents are also potent inhibitors of the enzymes used in target amplification processes or in many hybridization detection methods or, in the case of chaotropes, may interfere with hybridization itself. Therefore, it has been necessary to use additional steps to remove these agents and recover the nucleic acids.
The most commonly used procedure is to precipitate the nucleic acids from the sample using various salts and ethanol. The sample must be kept at reduced temperature (usually −20° C. or lower) for some hours and centrifuged at high speed in order to achieve good yields of nucleic acids in most instances.
Because other macromolecules also precipitate under these conditions producing a sticky, intractable mass that entraps the nucleic acids, it has been frequently necessary to resort to extraction of the sample with hazardous organic solvent mixtures containing phenol, cresol, and/or chloroform prior to ethanol precipitation. In some cases when anionic detergents are used, proteases that are active in the presence of these detergents, such as proteinase K or pronase, are used to partially degrade protein components of the sample to minimize entrapment during organic solvent extraction, and/or degrade components that may not be extracted by the solvent treatment.
It will be readily appreciated that these methods are complex, tedious, labor-intensive, and slow. If great care is not taken in performing the procedure, residual contamination with nucleases can occur, and the sample nucleic acids will be degraded or lost. Diagnostic tests performed with such samples may give false negative results. False negative results can also be obtained if residual anionic detergents, chaotropic salts, or ethanol remain in the sample and inhibit hybridization and/or target amplification procedures. If anionic detergents and proteases have been used, residual proteolytic activity can also degrade the enzymes used in target amplification and/hybridization detection reactions and produce false negative results. On the other hand, improper processing with these methods can also result in the isolation of denatured proteins or other macromolecules that can entrap labelled probes and produce false positive results with diagnostic tests involving nucleic acid hybridization. Thus, these procedures are not well suited for routine processing of biological specimens received in clinical laboratories in any quantity.
Particularly, trouble is encountered with many biological samples in which the desired nucleic acid species is RNA, and the sample contains significant amounts of RNAse of the “pancreatic” type (also frequently referred to as “ribonuclease A”). Pancreatic RNAses are present in serum and plasma and in many tissues of the body. They are resistant to denaturation by heat and acids and will even withstand boiling in 1 N HCl for 10 minutes without loss of activity. They are inhibited by anionic detergents, chaotropes, and organic solvents such as phenol, but are not irreversibly inactivated by these agents; therefore, when the detergents, chaotropes, or solvents are removed, the RNAse (if not eliminated by careful extraction) can proceed to degrade the desired RNA.
Exposure to strong alkali will irreversibly inactive these RNAses; however, such conditions also result in the degradation of RNA itself.
The present invention addresses these problems by providing a method for conveniently inhibiting and inactivating nucleases in biological samples while making available sample nucleic acids for hybridization assays, target amplification procedures, or other uses. Inhibitory detergents or chaotropes are not required in the sample, and there is no residual proteolytic activity. The method is simple and applicable to processing large numbers of samples simultaneously. Unlike ethanol precipitation methods, it does not use hazardous organic solvents, nor require equipment for cooling the sample or recovering precipitates by centrifugation.
SUMMARY OF THE INVENTION
The present invention features a procedure for irreversibly inactivating endogenous nucleases in biological samples by reducing the pH below that at which the endogenous nucleases present in the sample are active, adding a protease which is active at that pH and which degrades any nucleases that have not been irreversibly inactivated by exposure to low pH, and then inactivating the protease (after it has done its work) by raising the pH. At the higher pH, the chosen protease is either inactive or is irreversibly inactivated. If possible, the protease is chosen so as to aid in the digestion of other macromolecules in the sample that may interfere with the intended use of the sample, and chosen to help make available the desired nucleic acids by degrading microorganism cell walls, virus particles, ribosomes, and/or other structures containing the desired nucleic acids. Alternatively, solubilization of these structures and release of the nucleic acids may be effected by the use of detergents, heat, or other means once sample nuclease activity has been effectively controlled, reduced, or eliminated.
In general, the biological sample is adjusted to a low pH where the endogenous nucleases are either irreversibly inactivated or are effectively inhibited. Exogenous acid protease (such as pepsin) is then added or may be added simultaneously with the pH lowering solution. The action of the acid protease digests the endogenous nucleases present in the sample and irreversibly inactivates them so that they will not degrade the sample nucleic acids when the pH is subsequently raised. I
Kacian Daniel L.
Nunomura Kiyotada
Cappellari Charles B.
Gen-Probe Incorporated
Heber Sheldon O.
Myers Carla J.
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