What are the techniques in Molecular Biology?

Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.

Importance of techniques in Molecular Biology

Molecular biology focuses on the molecules of life, primarily nucleic acids and proteins. Cell biology is concerned with how these molecules are used by the cell to survive, reproduce, and carry out normal cell functions. Molecular techniques utilized in biomedical research to learn more about how cells function normally and how disruptions in this activity lead to disease. DNA sequencing and synthesis, chromosome integration, molecular hybridization, cellular screening, Polymerase chain reaction (PCR), DNA cloning, cellular culture, gel electrophoresis,  blotting, and molecular probing with DNA and protein arrays are the most frequently utilizing techniques in molecular biology for these studies.

Importance in disease diagnosis

A molecular diagnostic is a group of techniques that employ molecular biology principles to assessing biological markers in the organism’s genome and proteome, and how cells express their genes as proteins. Molecular diagnostics is utilized mainly in medicine to diagnose and monitor disease, detect risk, and determine which medications will be most effective for specific individuals.

Molecular diagnostic procedures are low-cost diagnostic methods that are very sensitive and specific. For the identification and epidemiological investigations of human viral infections, immunological-based approaches have been widely used.

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:

Denaturation

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.

Annealing

The temperature is decreased, and the primers attach to complementary DNA sequences.

Extension

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

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.

Blotting methods

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

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.

Steps

1. Preparation of samples or DNA extraction.
2. Amplification of target DNA sequence using PCR.
3.  Amplicon purification.
4. Pre-preparation for sequencing.
5. Sequencing of DNA.
6. 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.

Context and Applications

This topic is significant in the study of techniques in molecular biology and points out their importance, applications. Knowledge of available techniques allows students to identify which techniques are essential in the fields of medicine, biotechnology, and genetics and also useful in the professional exams of school, graduate, and post-graduate levels especially for Bachelors in Biology / Biotechnology and Masters in Biology /Biotechnology.

Practice Problems

Question 1: Northern blotting is employed for ______.

1. Identifying specific RNA
2. Identifying specific DNA
3. Identifying RNA & DNA
4. Identifying specific protein

Answer: Option 1 is correct.

Explanation: For DNA detection, southern blotting is used, and for RNA detection, northern blotting is used and proteins are detected using western blotting.

Question 2: The polymerase chain reaction is the_________ reaction.

1. DNA amplification
2. DNA degradation
3. DNA sequencing
4. None of the above

Answer: Option 1 is correct.

Explanation: Polymerase chain reaction is a technique for rapidly creating copies of DNA in billion numbers, allowing amplification of a small amount of DNA or a portion of it to a large enough amount to investigate in depth.

Question 3: Taq polymerase is used as _________ in PCR.

1. Primer
2. DNA Polymerase
3. Both 1 and 2
4. None of the above

Answer: Option 2 is correct.

Explanation: Taq polymerase, isolated from thermophilic bacteria named Thermus aquaticus, is used as the DNA polymerase enzyme.

Question 4: The determining step in PCR is ______.

1. Denaturation
2. Annealing
3. Extension
4. None

Answer: Option 1 is correct.

Explanation: Denaturation is critical that the temperature be kept at a high level for a long enough time to make sure that the DNA strands have completely separated.

Question 5: DNA sequencing is the technique for determining the exact sequence of bases in a DNA molecule in the laboratory.

1. True
2. False
3. May be true
4. None of the above

Answer: Option 1 is correct.

Explanation: The sequencer machine reads the lengthy chain of DNA in a sequential manner.