Definition of terms
- HAT – Hypoxanthine Aminopterin Thymidine
- Myeloma cells – Long-lived immune cells
- Aneuploidy – Implies an abnormal number of chromosomes
- MALDI-TOF – Matrix-Assisted Laser Desorption Ionization – Time of Flight
- SDS – Sodium Dodecyl Sulfate
- DNA – Deoxyribonucleic acid.
The cell culture technique has been one of the major tools in biological scientific research. The technique is common because it is simple to perform and the results obtained are easy to interpret. An artificial environment is modified to have physiological conditions like in vivo environment. Breakthroughs in tissue and cell cultures have to led to major developments in biomedical research. Cancer research has benefited a lot from advancements in cell and tissue cultures. Scientists utilize cell cultures to study the properties of cancer cells. In addition, drugs that are targeted on cancer cells are introduced to the cells in vitro. If the drugs show good efficacy levels in vitro, then they are taken to the next phase of testing which involves the use of humans.
Pandey (89) states that assessing the morphology of the cells is the first step in determining the development of cells in cell culture experiments. Different cell lines have different observable characteristics. The characteristics help to differentiate normal cells from abnormal cells. It is easy to visualize and characterize the morphological changes of the cell. However, it is difficult to characterize the causative agents for the changes. It is also difficult to characterize and measure the rate of change in the cell structure and morphology. Microscopy is the gold standard for assessing the progress of cell culture. Failed cell culture experiments contain cells that have different morphological characteristics that are different from those of the normal cells. The changes in the patterns of growth could be used to identify the cells with abnormal growth and development in cell culture experiments. Staining is essential in counting and conserving live cells. Some experiments are used to identify the causes of toxicity in cell culture experiments (Pandey 89).
The cell counting process is essential in identifying the growth rate of cells based on their external morphological features. Cell culture assays require an individual to identify the number of cells present in an experiment. Failure to identify the number of cells may result in the poor interpretation of results. It has been demonstrated that modifications of the growth media may increase or decrease the growth rate of cells in cell culture experiments (Pandey 89).
One of the commercialized techniques of cell culture is the monoclonal antibody production by hybridoma technique. Hybridoma involves secreting B cells from the immune system and fusing them with myeloma cells. In the laboratory, the process involves using modern molecular biology techniques in obtaining B-cells from the mouse and fusing them with myeloma cells. The process is conducted in an external environment that mimics the physiological conditions of the mouse, in the presence of HAT, a selective medium that is used to differentiate fused B-cells from unfused B-cells (Pandey 93).
The processes of cell culture are cumbersome, slow, and time-consuming. For example, the chances of success of hybridoma technology are low. In addition, the technology requires a high number of B-cells to increase the chances of success. The challenges of cell cultures have necessitated the development of molecular biology techniques. Molecular biology techniques are validated by comparing results from similar techniques conducted by independent researchers. Pandey (90) argues that activities in cell culture techniques enable researchers to manipulate the experimental designs to use molecular biology tools. The molecular biology techniques are easy to perform and results are obtained faster than in cell culture experiments (Pandey 90).
The biochemical and immunological assays in cell culture require maximum sterility. This is not the case when conducting similar assays using molecular biology tools and techniques. Molecular biology techniques are likely to replace cell cultures in the 21st century in clinical microbiology laboratories. The major molecular biology techniques applied in microbiology laboratories are microarrays, Edman sequencing, mass spectrometry, and proteome mining. These techniques are useful in characterizing protein complexes, protein profiles, protein arrays, and phosphorylation analysis by MALDI-TOF. The major challenge of the molecular biology techniques is that analysis of low-abundant proteins is not possible because proteins cannot be amplified like DNA. Moreover, there is a limitation of study of proteins since they are sensitive. Thus, the techniques could only be used on small scale. Combining molecular biology techniques with other complementary technologies leads to improved approaches to protein studies (Graves and Timothy 40).
The genomics approach in the microbiology laboratory will involve the acquisition of sequence data. For example, acquisition of complete genome sequence of Haemophilus influenza and Drosophila melanogaster. The genomic information of the two organisms was acquired from analyzing sequences of about 500-800 base pairs that were assembled to form a contiguous genome sequence. Graves and Timothy (41) state that the analysis of the genome sequence helps to locate the genes, control sequences and other features. The analysis is followed by various experiments which aim to decipher the functions of unknown genes.
Bioinformatics is another approach that is useful in molecular biology studies. Bioinformatics involves the use of computers to conduct post-genomics research. It involves computerized assembly of contigs, prediction of gene function, gene sequence examination, and storage of data generated in genome projects (Graves and Timothy 43).
According to Graves and Timothy (43), Edman sequencing involves the utilization of labeled amino-terminal sequences to form a peptide. Introduction of charges to the amino terminals generates derivatives that form stable amino acid groups which could be identified through electrophoresis. The approach is useful in characterizing membrane proteins. During electrophoresis, proteins are linearised with the help of SDS which disrupts the bonds holding polypeptides together. In the process, they gain negative charges equivalent to their masses. Edman sequencing begins at the N-terminus of a degraded protein and cannot proceed when there is a modification of the initial N-terminus. To overcome the challenge, scientists have developed mixed-peptide sequencing. The proteins from electrophoresis are transferred to a polyvinylidene difluoride platform for blotting. This allows extra experimental procedures in protein studies which culminate in obtaining more data for analysis (Graves and Timothy 44).
Mass spectrometry provides protein structural information. The information could include amino acid sequence and peptide masses. For example, the table below gives the masses of individual amino acids that help to determine peptide masses.
Mass spectrometry characterizes proteins based on the molecular mass of each peptide in protein molecules (Graves and Timothy 47). Protein identification is achieved by comparing peptide masses to online database protein or nucleotide data (Graves and Timothy 45). The process of protein identification is simplified through the use of the spectrum that equates and aligns each molecule in the peptide. The stages of sample processing are essential because they determine the quality of the output in mass spectrometry analysis. With an analytical approach, it is possible to identify human hemoglobin from an unknown sample of the human species. Tryptic digestion of the human hemoglobin yields 14 tryptic peptides. The VGAHAGEYGAEALER peptide has an exact monoisotopic mass of 1528.7348 Daltons. If the peptide is searched against mouse and human protein, then the SWISS-PROT database would give results similar to mouse hemoglobin (Graves and Timothy 47).
A microarray technique is a method of determining gene expression levels. Microarray techniques help to assess the messenger RNA (mRNA) abundances in different biological samples. The technique involves the use of solid support on which DNA molecules of known sequence are deposited (Pandey and Matthias 837). The DNA sequences (genes) in the microarray express different amounts of gene products. Probes in DNA microarrays have fluorophore which gives fluorescence when illuminated (Graves and Timothy 52). The intensity of fluorescence is directly proportional to the number of genes expressed in the biological sample. DNA microarray is useful in analyzing many gene samples to determine their expression levels. Microarray experiments yield a large amount of information which requires molecular sequence databases for analysis. The major limitations of microarray are high costs, unreliability, and complex approaches for validating findings (Graves and Timothy 52).
The ultimate goal of adopting molecular biology techniques is to improve the microbiology experiments so that they would generate more information. Molecular biology techniques help to incorporate other biological methods for further analysis like bioinformatics. Through this, a large amount of information is obtained from a molecular biology assay. Most of the molecular biology techniques are fast, comprehensive, and flexible (Pandey and Matthias 839). However, it has been shown that molecular biology techniques require expertise. If there are errors in the initial stages, then they would lead to misinterpretation of the results. Molecular biology tools are essential in identifying all the test sample proteins and their functions. Moreover, researchers are able to study protein structures and morphologies in both healthy and unhealthy states. Indeed, molecular biology techniques will eventually replace cell cultivation in the 21st-century clinical microbiology laboratory.
Graves, Paul R., and Timothy AJ Haystead. “Molecular biologist’s guide to proteomics.” Microbiology and Molecular Biology Reviews 66.1 (2002): 39-63. Print.
Pandey, Akhilesh, and Matthias Mann. “Proteomics to study genes and genomes.” Nature 405.6788 (2000): 837-846. Print.
Pandey, Shivanand. “Hybridoma technology for production of monoclonal antibodies.” Hybridoma 1.2 (2010): 88-94. Print.