Techniques used in molecular biology
Polymerase chain reaction
In 1983, Kary Mullis, an American scientist, devised a polymerase chain reaction. Polymerase chain reaction (PCR) is known as a technique for rapidly creating copies of a particular DNA sample in billions, permitting the amplification of a small amount of DNA or a portion of it to a large amount to investigate in depth.
Many processes used in genetic testing and research, including infectious agent identification and DNA sample analysis, rely on it. Using the PCR amplification technique, copies of very small amounts of DNA sequences are efficiently increased by a chain of temperature adjustments.
PCR technique is dependent on thermal cycling. Thermal cycling involves alternative heating and cooling of reactants to allow temperature-dependent processes such as the melting of DNA and enzyme-mediated DNA replication. PCR uses two major reagents: DNA polymerase and primers. Primers are oligonucleotides that are short single-strand DNA fragments that have complementary sequences to the target sequence of DNA. The three steps in PCR are:
In denaturation, the two strands of the DNA should be split, much like in DNA replication. The hydrogen bonds within the complementary DNA strands are broken as the temperature of the mixture is raised, causing the separation. It is the most essential step in PCR.
The temperature is decreased, and the primers attach to complementary DNA sequences.
The two strands of DNA are used as templates by the enzyme DNA polymerase, which is used to create a new DNA strand from nucleotides. Taq polymerase, obtained from Thermus aquaticus– thermophilic bacteria, is used as the DNA polymerase enzyme. The nucleotide sequence in the original template DNA strand determines the order in which the free nucleotides are inserted.
Because most PCR techniques require significant amounts of DNA, the cycle is repeated numerous times, typically 20–30. Obtaining a billion copies takes only 2–3 hours.
PCR helps to modify DNA fragments and helps in genetic marker analysis employed in forensic applications, mapping of hereditary features, and paternity testing. The study of gene expression and identification of harmful pathogens in high sensitivity.
Molecular cloning is referred to as a set of experimental methods in molecular biology to assembling recombinant DNA molecules and directing their replication in host organisms. Cloning is referred to as a technique that involves the reproduction of a single molecule to synthesize a population of cells with identical DNA molecules. In most cases, DNA sequences from two different organisms are used for molecular cloning, the source of the DNA to be cloned, and the species that serve as the living host for recombinant DNA replication.
Because of the use of molecular cloning procedures, screening assays for viral infection used prior to blood donation were developed quickly, dramatically lowering the occurrence of post-transmission of chronic viral diseases. The molecular tests for detection, quantification and virus characterization were aided by cloning and viral genome sequencing. For the development of vaccines, molecular cloning technology is becoming significant in the production of recombinant antigens.
Edwin M. Southern invented the technology for the analysis of DNA in 1975. Blotting methods are an auxiliary to gel electrophoresis, a method for separating DNA, RNA, and proteins with extraordinary resolving power, which is used to detect specific biomolecules in complex samples.
It is used for biomolecules that can bind their corresponding ligand while adhering securely to a support material like nitrocellulose, nylon, or paper membrane. Before being transferred to the membrane, the biomolecules of interest are usually sorted by size and/or charge. There are three main types of blotting techniques. For DNA detection, southern blotting is used, and for detection of RNA, northern blotting is used and proteins are detected using western blotting.
All of the procedures contain a step in which molecules are moved from the gel to a porous membrane, which is usually performed by soaking a solution across the gel and membrane with absorbent paper. Molecular hybridization with labeled nucleic acid probes detects specific sequences in the membrane for DNA and RNA. Antibodies that have been labeled are used to detect the proteins.
Blotting method applications are:
- Northern blotting allows researchers to compare the expression patterns of a gene across tissues, organs, stress conditions, pathogen infection and developmental stages.
- The approach has been used to show over expression of oncogenes and down regulation of tumor-suppressor genes in malignant cells.
- Southern blotting used in detecting RFLPs involved in the formation of genomic maps and used to identifying restriction fragments that contain DNA sequence.
- Western blotting is used for identification of tagged proteins, protein level changes at different time points and protein’s phosphorylation state.
DNA sequencing is the technique for determining the exact sequence of bases in a DNA molecule in a laboratory setting. The information a cell needs to assemble protein and RNAs is contained in the base sequence of DNA. As part of the human genome project, DNA sequencing technology was made faster and less expensive.
It is essential to research a gene’s nucleotide sequences in order to comprehend its structure and functions. The sequencer machine reads the lengthy chain of DNA in a sequential manner.
- Preparation of samples or DNA extraction.
- Amplification of target DNA sequence using PCR.
- Amplicon purification.
- Pre-preparation for sequencing.
- Sequencing of DNA.
- Analyzing data.
DNA sequencing is used to discover variations in a gene that may cause disease and make use in medicine for different purposes, including disease diagnosis and therapy, epidemiology investigations, and evolutionary studies between species or populations.
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