Reverse sample genome probing

Details Reverse sample genome probing Reverse sample genome probing

1991-06-21

2002-04-23

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C436S057000, C436S063000, C436S501000

Reexamination Certificate

active

06376173

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acid probes for the detection of microorganisms. More particularly, the invention relates to a “reverse probe” nucleic acid probe.
BACKGROUND OF THE INVENTION
Nucleic acid probes for the detection and analysis of microorganisms are known, although clinical applications of this technology are still comparatively rare.
These probes have clear potential value in the microbiology labs utilized in such disciplines as medicine, environmental and/or water resources engineering, agronomy and veterinary medicine. Certain probes and procedures have been developed for detecting the presence of microorganisms such as
Chlamydia trachomatis, Haemophilus ducreyi, Mycobacterium
and
Escherichia coli
, in clinical samples, environmental samples and the like.
A detailed review of gene probe technology is provided in the textbook “Gene Probes for Bacteria” (A. J. L. Macario and E. Conway de Macario, editors. Academic Press Inc. 1990. ISBN. 0-12-463000-6).
As a general overview, conventional gene probe techniques involve:
(a) selection of a nucleic acid probe (which may be based on the total nucleic acid of the species of interest; or some limited sequence thereof);
(b) affixing the nucleic acid from a sample onto a solid matrix (such as a nitrocellulose membrane);
(c) contacting the nucleic acid probe with the sample nucleic acid affixed on the matrix under conditions which permit hybridization; and
(d) detection of hybridization (as hybridization is regarded as evidence of the presence of the probe species within the sample).
Two of the most well known procedures used to probe microorganisms are:
(a) Southern blot (in which the sample nucleic acid is isolated, purified, subjected to restriction endonuclease digestion, subjected to electrophoresis, denatured, affixed to a solid matrix, then hybridized while affixed to the matrix with a radioactively-labeled probe); and
(b) dot or spot blot (in which lyzates of the sample, containing denatured nucleic acid, are affixed to the solid matrix without prior electrophoresis).
Thus, it will be apparent that the prior-art techniques require that the total nucleic acid from the sample be affixed to the matrix, in order to subsequently utilize a species specific probe. This procedure is very time consuming if it is desired to use a plurality of gene probes to attempt to characterize a sample (as a separate incubation is required for each gene probe employed).
The present invention mitigates certain of the disadvantages of the prior-art probe technology by reversing the sample/probe relationship.
SUMMARY OF THE INVENTION
Novel methods and compositions are provided for detecting the presence of a microorganism or a virus in a sample using nucleic acid isolated from the sample as a reverse genome probe. The method involves the steps of hybridizing the sample nucleic acid with a plurality of separately indentifiable nucleic acid standards prepared from organisms chosen based upon the origin of the sample, freeing the complexes of unbound sample and reagents, and detecting the complexes by means of a label attached to the sample nucleic acid. The indentity of the organism is obtained from the standard to which the sample nucleic acid hybridized. Optionally, the sample may be grown prior to analysis to increase the number of organisms. The method finds use in analyzing microorganism populations in environments where the microbial flora is poorly characterized.
Thus, in the broadest aspect of the present invention, a new and convenient method for the determination of the presence of more than one selected standard within a total microorganism sample is provided. In particular, the present invention provides:
Claim
1
In another embodiment of this invention, there is provided:
Claim to Master Solid Surface.
It will be appreciated that a “total” nucleic acid sample (i.e. a sample of the nucleic acid of the combined microorganisms present in the total sample) is employed as a reverse genome probe in the method of this invention.
As a further note of clarification regarding the difference between prior art gene probes and this invention, it will be recognized that the prior art probes may be prepared at and distributed from a central location (i.e. so that an end user provided with the probe then needs to prepare a filter or “solid surface” in order to complete the prior art probing process) whereas this invention permits the preparation of a master solid surface at a central location (so that an end user provided with a master solid surface needs only to prepare the reverse probe in order to undertake the process of this invention).
DETAILED DESCRIPTION
In accordance with the subject invention, methods and compositions are provided for indentifying a organism in a sample, particularly in a field sample from an uncharacterized microbial environmental such as an oil field. The presence of an organism may be detected using nucleic acid prepared from the sample itself as a reverse gene probe.
This invention is generally suitable for gene probing a wide variety of prokaryotic microorganisms, including bacteria and eukaryotic microorganisms, including fungi, as well as viruses. As will be readily recognized by those skilled in the art, different types of gene probes have different sets of advantages and disadvantages. Although this invention provides a means to conveniently provide an initial characterization of a complex specimen or sample, this convenience may come at a cost of not being able to distinguish between microorganisms having very similar genomes (i.e. this invention does not allow one to completely distinguish between species having strongly cross-hybridizing genomes. However, where closely related organisms with homologous cross-hybridizing genomes may be present in the sample, and it is desired to obtain more specific information, the technique may be combined with other techniques known to those skilled in the art to identify more specifically any closely related organisms.) Accordingly, the term “selected standard” as used herein includes both:
(a) a microorganism having a genome that doesn't cross-hybridize with any other; and
(b) a group of microorganisms having homologous, cross-hybridizing genomes (i.e. such a group represents a single selected standard).
In other words, a given selected standard may simply be one microorganism or it may be a group of closely related microorganisms. Thus, a given selected standard is different from another selected standard if they have genomes with little or no cross-hybridization.
In spite of this limitation, it will be readily apparent that the present invention provides a rapid means for the initial characterization of a sample, because only a single incubation (rather than multiple incubations) is required after a suitable master filter has been prepared.
This is because the “reverse probe” used in this invention is prepared using the total nucleic acid from the sample (whereas the probe of the conventional prior art technique is prepared with nucleic acid from the selected or target species). From this, it follows that the master solid surface used in this invention has affixed thereto spots of nucleic acid from selected standards of interest (whereas, as noted above, the prior art techniques involve the use of a solid surface having affixed thereto total nucleic acid from the sample and hence require an incubation with each conventional probe being utilized).
Additionally, it has been surprisingly discovered that the total specimen nucleic acid (i.e. nucleic acid obtained directly from the sample, without first isolating the species therein) is suitable for use as a “reverse genome probe”.
As noted above, the method of this invention generally involves three main steps:
1. Preparation of a Master Solid Surface.
2. Preparation of a Reverse Genome Probe.
3. Contacting the Reverse Genome Probe with the Master Solid Surface under conditions which permit hybridization.
Each of the above steps is described in detail below.
Master Solid Surface
The mast

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