Molecular biology is mainly concerned by understanding the methods responsible for the transmission and expression of the genetic information which ultimately manages cell structure and function. Comprehending the molecular biology of cells is basic to all biological sciences, due to its growing number of practical applications in biotechnology, agriculture and medicine.
Meaning of Molecular Biology:
The word molecular biology was first employed in the year 1945 by William Astbury to refer to the study of the physical and chemical structure of the biological molecules. Contemporary molecular biology is the study of life at the molecular level; and it is mainly concerned by the understanding and interactions among the different systems of a cell, comprising the interactions between the different kinds of RNA, DNA and protein biosynthesis and how such reactions are regulated - the methods responsible for the transmission and expression of the genetic information which finally governs cell structure and function that is, the study of the procedure of replication, transcription and translation of the genetic material. The central dogma of molecular biology where the genetic material (that is, DNA) is transcribed to RNA and then translated to protein gives the starting point for comprehending the field.
Relationship by Other Biological Sciences:
Molecular signs of evolution exhibit that evolutionary relationships between species are reflected in their DNA and proteins - in their genes and gene products. Researchers in the molecular biology employ methods and ideas from other regions of chemistry and biology specifically genetics - the transfer of biological information from cell to cell, from parents to offspring and therefore from generation to generation and to the consequences on organisms and biochemistry- the study of the chemical substances and important procedures occurring in living organisms specifically the role, the function and the structure of biomolecules.
Much of the work in molecular biology is quantitative and need ideas from computer science in bioinformatics and computational biology. Molecular genetics - the study of gene structure and function is a well-known sub-field of molecular biology.
Most of the other areas of biology focus on molecules, either directly studying their interactions in their own right like in cell biology and developmental biology or indirectly where the methods of molecular biology are employed to infer historical attributes of populations or species as in the fields of evolutionary biology like population genetics and phylogenetic.
Techniques of Molecular Biology:
To understand various biochemical events of prokaryotic and eukaryotic cells at molecular level and be capable to characterize, isolate and manipulate molecular components of cells and organisms, a broad array of bio-physico-chemical methods is employed in the molecular biology. These comprise:
1) Expression of Cloned Genes:
Molecular cloning lets the determination of the nucleotide sequences of genes and as well provides new approaches to get large amounts of proteins for structural and functional feature. In expression colony, DNA coding for a protein of interest is cloned by using polymerase chain reaction or restriction enzymes into a plasmid or phase vector termed as expression vector.
The plasmid can be inserted to either bacterial or animal cells; DNA coding for a protein of interest is now in a cell and the protein can now be expressed. A diversity of systems, like inducible promoters and specific cell-signaling factors, are vailable to aid express the protein of interest at high levels.
Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity beneath a diversity of situations, the protein might be crystallized so its tertiary structure can be studied, or in the pharmaceutical industry, the action of new drugs against the protein can be studied.
2) Polymerase Chain Reaction:
Molecular cloning lets individual DNA fragments to be propagated in the bacteria and isolated in huge amounts. The polymerase chain reaction is a versatile method for copying DNA, and lets a single DNA series to be copied repeatedly or modified in predetermined ways; necessarily it is employed for repeated replication of a defined segment of DNA. Single DNA molecules can therefore be amplified to yield readily detectable quantities of DNA which can be isolated and quantitatively measured.
3) Gel Electrophoresis:
Gel electrophoresis is one of the main tools of molecular biology. It is a general process in which molecules (that is, DNA, RNA and proteins) are separated based on the rates of their migration in an electric field. A gel, generally made from agarose or polyacrylamide, is positioned between two buffer compartments having electrodes. The sample is then pipette into preformed slots in the gel, and the electric field is turned on; the gel acts similar to a sieve, selectively retarding the movement of larger molecules.
Smaller molecules thus move via the gel more quickly, letting a mixture of nucleic acids to be separated on the basis of size.
4) Nucleic Acid Hybridization:
Cloning facilitated the isolation and classification of individual genes. Though, understanding the role of genes in cells, needs analysis of the intracellular organization and expression of individual genes and their encoded proteins. Nucleic acid hybridization is a method for detecting and analyzing series of homologous DNA. This facilitates the mapping of genes, to chromosomes, the analysis of gene expression, and the localization of proteins to sub cellular organelles. In this manner, it is possible to study genetic differences among organisms or individuals.
Hybridization can be accomplished by southern or northern blotting.
Southern blotting: Southern blotting is a process for probing for the presence of a particular DNA sequence in a DNA sample and it lets a researcher to find out not only whether a specific sequence is present in a sample of DNA, however how many such sequences there are; and the size of the restriction fragments which encompass these sequences.
Northern blotting: Messenger RNA can as well be subjected to hybridization analysis, in an analogous process termed as Northern blotting employed to study the expression patterns of a specific kind of RNA molecule and is basically a combination of denaturing RNA gel electrophoresis and a blot.
5) Restriction Fragment Length Polymorphism Analysis:
DNA fragments which outcome from cutting a specific piece of DNA by a specific restriction enzyme provides a feature pattern of bands on gel electrophoresis. Fact band corresponds to a DNA restriction fragment of a specific length. Such difference are termed as restriction fragment length polymorphisms (RLFPs) serving as genetic marker for a specific location the genome. A given FLP marker often takes place in many variants in a population and is inherited in a Mendelian fashion. Genetic markers are employed for making linkage maps. RLFP analysis is significant in the diagnosis of genetic disorders and in the forensic applications.
Escherichia coli as Experimental Model:
Due to their comparative simplicity, prokaryotic cells (bacteria) are ideal models for studying numerous basic aspects of biochemistry and molecular biology. The most methodically studied species of bacteria is E. coli that is the most favored organism for investigation of the basic methods of molecular genetics. Most of the present theories of molecular biology: DNA replication, the genetic code, gene expression and protein synthesis - are derived from the studies of E. coli.
E. coli is helpful to molecular biologists as its relative simplicity and easiness of its propagation and study in the laboratory. For illustration the genome of E. coli comprises of around 4.6 million base pairs and encodes around 4000 different proteins. Whereas the human genome is more complicated with around 3 billion base pairs and encodes around 100,000 different proteins. The small size of E.coli genome gives benefits for genetic analysis and the series of whole E.coli genome has been find out.
Molecular genetic experiments are further facilitated through the fast growth of E.coli beneath well defined laboratory conditions. E. coli can divide each 20 to 60 minutes, based on culture conditions and a clonal population of E.coli all cells derived through division of a single cell of origin can be isolated as a colony grown on agar having medium. Bacterial colonies have many cells and choosing and analyzing genetic variants of E.coli strain is simple and rapid. This usually contributes to the success of experiments in molecular genetics.
E.coli can split rapidly in nutrient mixtures such as glucose, salts, amino acids, vitamins and nucleic acid precursors. Though, E. coli can also grow in much simpler media comprising of only salts as source of nitrogen (like ammonia) and a source of carbon and energy (like glucose). However in such simple medium, the bacteria grow a little slowly (that is, a division time of around 40 minutes) as they should synthesize all their own amino acids, nucleotides and other organic compounds.
The capability of E. coli to carry out such biosynthetic reactions in simple defined media has made them very helpful in explaining the biochemical pathways involved. Therefore, the rapid growth and simple nutritional needs of E. coli have very much facilitated basic experiments in both molecular biology and biochemistry.
However bacteria are models for studies of cell properties, they can't be employed to study features of cell structure and function which are unique to eukaryotes. Yeasts, the simplest eukaryotes, encompass a number of experimental benefits similar to those of E. coli and encompass provided a model for studies of numerous features of eukaryotic cell biology.
The genome of the most studied yeasts, Saccharomyces cervisae, comprises of 12 million base pairs of DNA and contains around 6000 genes; and is around 3 times bigger than that of E. coli it is much more manageable than the genomes of more complex eukaryotes, like humans. Yeasts can be readily grown in the laboratory and can be studied by most of the similar molecular genetics approaches that have proved successful by E. coli.
However yeasts don't replicate as fast as bacteria, they still split as frequently as every 2 hours and can simply be grown as colonies from a single cell. Yeasts can be employed for a diversity of genetic manipulations alike to those that can be performed by employing bacteria. Yeast mutants have been significant in comprehending many basic procedures in eukaryotes, comprising DNA replication, RNA processing, transcription, protein sorting and regulation of the cell division.
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