Structural Biochemistry/Methods for Determining Recombinant DNA in the Environment

A. Introduction

One of the most significant contributions of Recombinant DNA technology is the ability to manipulate the DNA of microorganisms. Also, referred to as Recombinant DNA (rDNA) technology, this manipulation of DNA involves the insertion of DNA segments from various organisms into a different host (rDNA host). This is often practiced through the use of a vector which allows for the DNA to replicate in the host cell producing a new manipulated microorganism. Thus, the practice of the manipulation of microorganisms has been the focus of hundreds of companies, laboratories, and even governmental agencies. The reason that the manipulation of DNA microorganisms is such a studied topic is the benefits it could provide on a economic level. Possible areas of the use of manipulated microbes include as a organism for degrading environmental contaminants, pesticides, providing protection for agricultural crops, in medicine, etc. The release of genetically engineered microorganisms (GEM) however has been a much disputed topic. The worries are the effects that would result from the addition of genetically altered microorganisms to the environment. The two main concerns are 1. host cells could contain pathogenic genes in the rDNA that was originally unknown which provides threats to all living organisms. 2. the altered organisms might have effects on the ecosystem especially in terms of evolution and providing an organism an advantage of another possibly altering such things as the nutrient cycle, energy flow, and ecosystem. This is the main reason that the addition of GEM to the environment (genetically engineered microorganisms) must be carefully studied and regulated. Research though therefore has been done therefore do identify genetically altered microorganisms in the environment led by three researchers from the University of Tennessee.

B. Requirements for rDNA Environmental Monitoring

In order for a technique method/applied to observe and search for genetically engineered microorganisms in the environment, a series of questions must first be asked to determine if a method is valid and/or a realistic technique.

1. It should be applicable under a wide variety of environmental conditions 2. It should be suitable for technical application in terms of its simplicity. 3. It should be able to detect, identify and enumerate the GEMs 4. It should be sensitive and specific to detect a small population 5. It should be capable of differentiating the specific GEMS from the other organisms in the environment 6. It should be able to discriminate GEMS from other strains of the same species 7. It should be efficient, cost effective, and time-economic.

These methods reflect the rules determined by the three scientists from the University of Tennessee

C. Conventional Methods

I. Selective Plating and Enrichment Techniques

The process of selective plating of microorganisms involves the presence of a selective media. The selective media can be made differential on basis of physical growth conditions (temperature, oxygen concentration) or constituents. In terms of detecting the rDNA of microorganisms, plating only selects for the rDNA host rather than a specific rDNA sequence. Complex media are used to accommodate the complexity of rDNA host which allows for the differentiation between the non recombinant strains. Furthermore, this technique is only considered a preliminary technique because it must be considered presumptive, which requires conformation for an rDNA to be present.

II. Enumeration by the Most-Probable-Number (MPN) Method

In this method, samples are first diluted to extinction and then viable cells are allowed to grow in tubes of appropriate medium. Then, a probability theory is performed to determine the original density of the population. The medium used determines both selection or enrichment and a species growth and activity thus it is another method of differentiation. There are some drawbacks however to this method which includes the necessity to use a large number of dilutions and tubes in order for precision.

III. Epifluorescence Count Technique

The technique involves concentrating the bacteria onto membrane filters, then staining the bacteria, and then counting the bacteria by microscopy. This technique know is not applied as much for GEM because of the fact that it lacks specificity like the other conventional techniques.

Weaknesses of the Conventional Method

1. weakness is that the technique lacks specificity. 2. technique require the growth of target organisms and consequently will underestimate or produce false-negative results if organisms are stressed at a immediate time of sampling. 3. there is no universal medium that allows growth of all potential host organisms within the sample.

D. Developing Methods

I. Immunological Techniques-The technique involves the use of antibodies (either polyclonal and monoclonal antisera) providing a sensitive and specific technique for identifying GEM.

II. Enzyme-Linked Immunosorbent assay (ELISA)- This biochemical technique involves the use of a particular antibody that is used to find a specific antigen, or vice versa in which a particular antigen is used to detect a particular antibody. This process is sensitive and specific, which might reflect its value when locating GEM (genetically engineered microorganisms).

III. Radioactive Markers-This technique involves the conjugation of some radioactive species, which allows for Genetically engineered microorganisms to be marked radioactively. The main drawback though is the cost and high amounts of radioactive use.

IV. Fluorescent Markers- This technique also involves the use of a antibody that is conjugated to a fluorescent dye. The antibody is added to a sample of interest and therefore could be observed by fluorescence microscopy.

V. Use of Plasmid Epidemiology and Restriction Profiles (DNA fingerprinting)-This technique is a method to study specific genetic engineered microorganisms by specifically studying the plasmid of the microorganisms. The plasmids are separated by agarose gel electrophoresis and studied by staining the gel by ethidium bromide and observing by ultraviolet light.

VI. Use of Selectable Genotypic Markers-Two different markers can be used to study GEM

1. chromogenic markers

2. antibody or heavy resistance markers-many of these markers are found in plasmids or transposons which can be used to incorporate into bacterial chromosomes.

The markers could be placed on either the plasmid or the chromosome of the microorganism.

VII. Use of nucleic Acid Sequence Analysis

VIII. Nucleic Acid Hybridization Techniques

IX. DNA: DNA Colony Hybridization

X. Southern Blot Hybridization

XI. Nucleic Acid Hybridization with DNA Extracts

XII. DNA: RNA Hybridization

XIII. Use of Biotinlyated Probes

REFERENCES Burlage R, Jain R, Sayler G. "METHODS FOR DETERMINING RECOMBINANT DNA IN THE ENVIRONMENT." Critical Reviews in Biotechnology, 8:1-33-84.