GENE PROBES

SOIL MICROBIOLOGY

BIOL/CSES 4684



This webpage was created by Katie Corbin and Charles Hagedorn



1. OVERVIEW OF GENE PROBE METHODOLOGY

A gene probe is defined as "a strand of nucleic acid that can be labeled and hybridized to a complementary molecule from a mixture of other nucleic acids" (169, Sylvia).  The method is one of the most basic nucleic acid hybridization techniques and is based on the structure of the nucleic acids and the genetic mechanisms that synthesize microbial compounds.  Gene probe analysis is used to identify the existence of specific organisms or specific sequences in a sample.  A known DNA sequence of a gene is targeted in the process.  The gene can be organism specific or may encode for the production of an enzyme unique to a metabolic pathway.  One example of this is identifying the enzymes used for N2 fixation.  The goal is to determine the presence or absence of any of the many types of nitrogen fixing microorganisms that contain these enzymes.

Once the target gene sequence is identified, then the complementary strand of nucleic acid (the gene probe) is constructed.  In order to detect if the gene probe combines with the bacterial DNA, specific nucleotides are labeled with either radioactive isotopes or nonradioactive chemicals (e.g. colorimetric or chemo- or bioluminescent). 

After the formation of the gene probe, the bacterial cells are lysed to release the DNA.  The DNA from the bacteria as well as the gene probes are then denatured to split the double strands into single strands.  The two are then allowed to intermingle and possibly hybridize.  If hybridization does occur, that is the indication that the targeted gene is in fact present. The degree of hybridization can be correlated with the numbers or biomass of organisms containing the targeted gene.   

The principles of gene probe analysis form the basis for many more sophisticated techniques in genetic analysis. Hybridization with a gene probe to detect a corresponding gene sequence can be performed against nucleic acids, individual cells, and colonies. The following diagram shows how a transgenic bacterium, containing a unique sequence from an insect, is created and the novel (or unique) gene sequence (gene A in the image) is then detected by a DNA probe that will hybridize only with gene A. This is how the "clone" (transgenic bacterium containing gene A) is selected and identified.



2. OVERVIEW OF GENE PROBE PROCEDURES
Procedures for nucleic acid probes can be grouped into three major categories based on strategies used to construct the probe: total genomic DNA, cloned restriction fragments, and synthetic deoxynucleotides.

With total genomic DNA hybridization, the entire total genomic DNA of one organism is used as a target for hybridization with the total genomic DNA of another organism. This procedure has been widely used in microbial systematics to determine relationships among closely related bacteria, but has not been very useful in detection and identification of organisms because preparation of the DNA and quantification of percent homology are time consuming and do not lend well to large-scale screening.

The construction of probes from restriction fragments of DNA cloned into plasmid vectors is the most widely used approach and involves both shotgun and targeted approaches. In the shotgun approach little is known about the resultant probe DNA other than size and performance as a probe. This approach is much less time consuming than the targeted approach, which involves identification and characterization of specific clones (this is the approach shown in the above diagram). One problem with the shotgun approach is that one may never clone a DNA fragment of desired specificity since chance ultimately determines the size of the cloned fragments. The first commercially available probe for detecting Salmonella in food was a shotgun probe. The targeted strategy involves cloning a particular genetic determinant and the cloned fragment is then used as a specific probe. These probes are also called function-specific probes because the function of the genetic determinants is usually known (e.g. nitrogenase) and the probes can be used as an indicator of metabolic activity.

Lastly, the recent development and simplification of DNA and RNA sequencing methods and automated DNA synthesis machines have made possible the designing of probes of defined sequence and specificity. Synthetic probes are generally very short, from 15 to 30 bases long, and can be less specific than restriction fragment probes because the instability caused by a mismatched pair is much higher for short hybrids than for long ones. Synthetic probes may also be less sensitive, due to their small size, because less label can be incorporated per molecule. Today, many of the rRNA probes used to study relationships between kingdoms of organisms (e.g. Archebacteria vs Eukaryote) are synthetic probes.

False positive results can be very common with probes if target sequences are not highly specific.  To minimize this, (1) use both negative controls (no target DNA) and positive controls (with target DNA), (2) always probe duplicates samples of each unknown or isolate, and (3) prepare probes very carefully. The following image shows the "Esprit" system from Life Science Resources that is used for 3-D visualization of probe hybridization and expression.



3. PROS AND CONS
The most important concerns with using DNA probe technology are first, the specificity of the DNA used in making the probe, and, secondly, how to relate hybridization results with some other parameter such as target population size or metabolic activities. The obvious problem is that probes could hybridize with both living and dead cells, as well as naked DNA. For the procedures described in Section 2 (above), the major advantage of using total genomic DNA probes is that cloning is not involved in the development of the probe while the obvious disadvantage of this procedure is the likelihood of cross-reactivity arising from homology between genes highly conserved in species.

Gene probes (usually function-specific cloned restriction fragment probes) have been widely used in population studies in microbial ecology and to evaluate the survival and fate of transgenic organisms released into the open environment. To test transgenic microbes for thier possible use in agricultural of bioremediation applications, it is essential to be able to identify the released organism to monitor its fate and survival. Gene probes provide one of the most reliable methods to do this.



4. ADDITIONAL SOURCES OF INFORMATION
Sylvia, D.M., J.J. Fuhrmann, P.G. Hartel, and D.A. Zuberer. 1998. Principles and Applications of Soil Microbiology. Prentice Hall, Rutgers, NJ. pp.169-173.

Levin, M.A., R.J. Seidler, and M. Rogul. 1992. Microbial Ecology: Principles, Methods, and Applications. McGraw-Hill, Inc. NY. pp.65-92.

Sorensen, A.H., V.L. Torsvik, T. Torsvik, L.K. Poulsen, and B.K. Ahring. 1997. Whole-cell hybridization of Methanosarcina cells with two new oligonucleotide probes. Appl. Environ. Microbiol. 63:3043-3050.



5. LINKS TO OTHER SITES ON GENE PROBES
Candida albicans This link provides access to the web's Virtual Genome Center and the C. albicans page on gene identification and probes developed for this fungus.

Molecular Biology Imaging This commercial site from Life Science Resources offers a wide variety of nonradioactive probes available for detecting gene expression in both live and dead cells.


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