Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Molecular Diagnostics
Conclusion

Any effective diagnostic test must be: 1) highly specific for the target molecule; 2) sensitive enough to detect small amounts of the target; and 3) simple enough to easily yield unambiguous results. There are two types of molecular diagnostic Methods: one is based on the affinity of an antibody for a specific antigen, and the other is based on the identification of specific nucleotide sequences using Hybridization or PCR.

The most common immunological method is ELISA. Briefly, it consists of the following steps: 1) immobilization of the sample on a solid support; 2) addition of a primary antibody specific to the target antigen and its binding to the target antigen; 3) addition of a secondary antibody–enzyme conjugate that binds to the primary antibody; 4) addition of a colorless substrate, which is converted into a colored compound by the enzyme within the conjugate. A color change in the reaction mixture indicates the presence of the target molecule in the sample.

ELISA is used to detect various Proteins, identify Viruses and Bacteria, and determine low-molecular-weight compounds in a wide range of biological samples. To increase the Specificity of primary Antibodies, Monoclonal Antibodies are often used for Diagnostics. To reduce costs, a technique of cloning their fragments in E. coli is employed to generate a combinatorial library, which is then used to obtain a wide range of Fv fragment combinations.

Hybridization is a highly sensitive and specific method for detecting nucleotide sequences in biological samples. It has been used to develop methods for identifying pathogenic microorganisms in clinical samples and various Microorganisms in the environment.

DNA diagnostics is based on the detection of known nucleotide sequences; for this purpose, specific primers are synthesized and the target sequence is amplified. This allows The Use of non-radioactive detection systems (e.g., chemiluminescent methods) or the detection of PCR products by gel Electrophoresis. Additionally, PCR products can be labeled with a fluorescent dye attached to the 5' end of the primer.

In forensic medicine, DNA fingerprinting is finding increasingly widespread application, based on the fact that each individual's DNA produces a unique pattern of hybridization bands. Human minisatellite DNAs, which do not encode any proteins and are highly variable, are typically used as probes.

To characterize plant DNA, a set of arbitrary oligonucleotide primers is used to perform PCR Amplification of random DNA fragments, followed by electrophoresis to obtain a DNA band pattern specific to each plant; this approach is known as RAPD.

DNA diagnostic methods are also used to detect point Mutations in a given Gene. One approach involves the ligation of two oligonucleotide primers. If There is a mismatch of even a single nucleotide at the junction of the hybridized oligonucleotides, ligation does not occur.

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Review Questions

1. Briefly describe how PCR can be used to detect Changes in the human ß-globin gene that lead to Sickle Cell anemia.

2. Explain THE PRINCIPLE OF the PCR/OLA method.

3. What is the ELISA method?

4. Describe three methods of non-radioactive DNA labeling. What are the advantages of non-radioactive detection methods?

5. You are tasked with developing a simple, sensitive, and reproducible test to detect a double-stranded DNA virus that causes a lethal infection in cattle. Since Treatment efficacy depends on early and accurate diagnosis, it is necessary to use methods that can detect the virus at its minimum level in the infected animal before any symptoms appear. Briefly describe and justify your course of action.

6. What do sensitivity, specificity, and simplicity mean in the context of diagnostic tests?

7. How is Chagas disease currently diagnosed? How can the existing Procedure be improved?

8. Why does the use of fluorescent Dyes facilitate the detection of specific nucleotide sequences?

9. WHAT IS A "molecular beacon" probe and how does it work?

10. What is DNA fingerprinting and how is it used to characterize trace amounts of DNA in forensic medicine?

11. What is the RAPD method and how is it used to identify genetic variants of crops?