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Molecular biology is the branch of
biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mechanisms, Development ...

biology
that seeks to understand the
molecular A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A molecule is an electrically neutral group of two or more atom An atom is the smallest unit of ordinary matter In ...

molecular
basis of
biological activity In pharmacology Pharmacology is a branch of medicine and pharmaceutical sciences concerned with drug or medication action, where a drug may be defined as any artificial, natural, or endogenous (from within the body) molecule which exerts a bio ...
in and between
cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Closed spaces * Monastic cell, a small room, hut, or cave in which a monk or religious recluse lives * Prison cell, a room used to hold peopl ...
, including
molecular A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A molecule is an electrically neutral group of two or more atom An atom is the smallest unit of ordinary matter In ...
synthesis, modification, mechanisms, and interactions. The study of chemical and physical structure of biological macromolecules is known as molecular biology. Molecular biology was first described as an approach focused on the underpinnings of biological phenomena - uncovering the structures of biological molecules as well as their interactions, and how these interactions explain observations of classical biology. In 1945 the term molecular biology was used by physicist William Astbury. The development in the field of molecular biology happened very late as to understand that the complex system or advantageous approach would be made in simple way of understanding by using bacteria and bacteriophages this organism yields information about basic biological process more readily than animal cell. In 1953 then two young men named Francis Crick and James Watson working at Medical Research Council unit, Cavendish laboratory, Cambridge, made a double helix model of DNA which changed the whole research scenario they proposed the DNA structure based on previous research done by Rosalind Franklin and Maurice Wilkins then the research lead to finding DNA material in other microorganisms, plants and animals. Molecular biology is not simply the study of biological molecules and their interactions; rather, it is also collection of techniques developed since the field's genesis which have enabled scientists to learn about molecular processes. One notable technique which has revolutionized the field is the polymerase chain reaction (PCR), which was developed in 1983. PCR is a reaction which amplifies small quantities of DNA, and it is used in many applications across scientific disciplines, as will be discussed later. The
central dogma of molecular biology The central dogma of molecular biology is an explanation of the flow of genetic information within a biological system. It is often stated as "DNA makes RNA, and RNA makes protein", although this is not its original meaning. It was first stated by ...

central dogma of molecular biology
describes the process in which DNA is transcribed into RNA, which is then translated into protein. Molecular biology also plays a critical role in the understanding of structures, functions, and internal controls within individual
cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Closed spaces * Monastic cell, a small room, hut, or cave in which a monk or religious recluse lives * Prison cell, a room used to hold peopl ...
, all of which can be used to efficiently target new
drug Uncoated tablets, consisting of about 90% acetylsalicylic acid, along with a minor amount of inert fillers and binders. Aspirin is a pharmaceutical drug A medication (also referred to as medicine, pharmaceutical drug, medicinal drug or s ...

drug
s, diagnose disease, and better understand cell physiology. Some clinical research and medical therapies arising from molecular biology are covered under
gene therapy Gene therapy is a Medicine, medical field which focuses on the genetic modification of cells to produce a therapeutic effect or the treatment of disease by repairing or reconstructing defective genetic material. The first attempt at modifying h ...

gene therapy
whereas the use of molecular biology or
molecular cell biology Cell biology (also cellular biology or cytology) is a branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, ...
in medicine is now referred to as
molecular medicine Molecular medicine is a broad field, where physical, chemical, biological, bioinformatics Bioinformatics () is an field that develops methods and s for understanding data, in particular when the data sets are large and complex. As an inter ...
.


History of molecular biology

Molecular biology sits at the intersection of biochemistry and genetics; as these scientific disciplines emerged and evolved in the 20th century, it became clear that they both sought to determine the molecular mechanisms which underlie vital cellular functions. Advances in molecular biology have been closely related to the development of new technologies and their optimization. Molecular biology has been elucidated by the work of many scientists, and thus the history of the field depends on an understanding of these scientists and their experiments. It all begins with the phenomenon of transformation in the bacteria,  in 1928, Frederick Griffith, observed a phenomenon of transformation from one bacterium to other ow known as genetic transformation At that time, he couldn't explain the phenomenon of transformation. Later in 1944, three scientists Oswald Avery, Colin Macleod and Maclyn McCarty, demonstrated the whole phenomenon of transformation in the bacteria. After, two years in 1930, molecular biology was established as an official branch of science. But the term “Molecular Biology” wasn't coined until 1938 and that was done by the scientist Warren Weaver, who was working as the director of Natural sciences at Rockefeller Foundation. From the following experiment it was concluded that DNA is the basic genetic material which caused the genetic changes. Basic composition of the DNA was known that it contains four bases known as – Adenine, Guanine, Thymine and Cytosine. So, on the bases of the chemical composition and the X-ray crystallography, done by Maurice Wilkins and Rosalind Franklin the DNA structure was proposed by James Watson and Francis Crick. But, before the Watson and Crick proposed the DNA structure, in 1950 Austrian born scientist Erwin Chargaff, proposed the theory / rule oday known as- Chargaff's rule which stated that the number of Adenine and Thymine and Guanine and Cytosine are in equal proportion. The Chargaff's rule "Chargaff's rule stated that DNA ''from any species of any organism should have a 1:1 stoichiometric ratio of''
purine Purine is a heterocyclic 125px, Pyridine, a heterocyclic compound A heterocyclic compound or ring structure is a cyclic compound that has atoms of at least two different chemical element, elements as members of its ring(s). Heterocyclic chemi ...

purine
''and pyrimidines (i.e., A+G=T+C) and, more specifically, that the amount of''
guanine Guanine () (symbol A symbol is a mark, sign, or word In linguistics, a word of a spoken language can be defined as the smallest sequence of phonemes that can be uttered in isolation with semantic, objective or pragmatics, practical meaning ...

guanine
''should be equal to''
cytosine Cytosine () (symbol A symbol is a mark, sign, or that indicates, signifies, or is understood as representing an , , or . Symbols allow people to go beyond what is n or seen by creating linkages between otherwise very different s and s. Al ...

cytosine
''and the amount of adenine should be equal to''
thymine Thymine () (symbol A symbol is a mark, sign, or word In linguistics, a word of a spoken language can be defined as the smallest sequence of phonemes that can be uttered in isolation with semantic, objective or pragmatics, practical m ...

thymine
''. This pattern is found in both strands of the DNA".'' The field of genetics arose as an attempt to understand the molecular mechanisms of
genetic inheritance Heredity, also called inheritance or biological inheritance, is the passing on of traits from parents to their offspring; either through asexual reproduction Asexual reproduction is a type of reproduction that does not involve the fusion o ...

genetic inheritance
and the structure of a
gene In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mecha ...

gene
.
Gregor Mendel Gregor Johann Mendel (; cs, Řehoř Jan Mendel; 20 July 1822 – 6 January 1884) was a meteorologist, mathematician, biologist, AugustinianAugustinian may refer to: *Augustinians Augustinians are members of Christian religious orders tha ...

Gregor Mendel
pioneered this work in 1866, when he first wrote the laws of genetic inheritance based on his studies of mating crosses in pea plants. One such law of genetic inheritance is the
law of segregation Mendelian inheritance is a type of that follows the principles originally proposed by in 1865 and 1866, re-discovered in 1900 by and , and popularized by . These principles were initially controversial. When Mendel's theories were integra ...
, which states that diploid individuals with two
allele An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is one of two, or more, forms of a given gene In biology, a gene (from ''genos'' "...Wilhelm Johannsen coined the word gene to describe the Mendelian_inheritance ...
s for a particular gene will pass one of these alleles to their offspring. Because of his critical work, the study of genetic inheritance is commonly referred to as
Mendelian genetics Mendelian inheritance is a type of biological inheritance Inheritance is the practice of passing on private property, titles A title is one or more words used before or after a person's name, in certain contexts. It may signify either ...

Mendelian genetics
. A major milestone in molecular biology was the discovery of the structure of DNA. This work began in 1869 by
Friedrich Miescher Johannes Friedrich Miescher (13 August 1844 – 26 August 1895) was a Swiss physician and biologist. He was the first scientist A scientist is a person who conducts scientific research The scientific method is an Empirical evidence, empi ...

Friedrich Miescher
, a Swiss biochemist who first proposed a structure called ''nuclein'', which we now know to be deoxyribonucleic acid, or DNA. He discovered this unique substance by studying the components of pus-filled bandages, and noting the unique properties of the "phosphorus-containing substances." Another notable contributor to the DNA model was
Phoebus Levene Phoebus Aaron Theodore Levene (25 February 1869 – 6 September 1940) was an American biochemist Biochemists are scientists who are trained in biochemistry Biochemistry or biological chemistry, is the study of chemical process In a scien ...
, who proposed the "polynucleotide model" of DNA in 1919 as a result of his biochemical experiments on yeast. In 1950,
Erwin Chargaff Erwin Chargaff (11 August 1905 – 20 June 2002) was an Austro-Hungarian-born American biochemist Biochemists are scientists who are trained in biochemistry Biochemistry or biological chemistry, is the study of chemical process In a scie ...
expanded on the work of Levene and elucidated a few critical properties of nucleic acids: first, the sequence of nucleic acids varies across species. Second, the total concentration of purines (adenine and guanine) is always equal to the total concentration of pyrimidines (cysteine and thymine). This is now known as Chargaff's rule. In 1953,
James Watson James Dewey Watson (born April 6, 1928) is an American molecular biologist, geneticist A geneticist is a biologist Francesco Redi, the founder of biology, is recognized to be one of the greatest biologists of all time A biologist is a ...

James Watson
and
Francis Crick Francis Harry Compton Crick (8 June 1916 – 28 July 2004) was a British molecular biologist, biophysicist, and neuroscientist A neuroscientist (or neurobiologist) is a scientist A scientist is a person who conducts Scientific method, scie ...

Francis Crick
published the double helical structure of DNA, using the
X-ray crystallography X-ray crystallography (XRC) is the experimental science determining the atomic and molecular structure of a crystal A crystal or crystalline solid is a solid material whose constituents (such as atoms, molecules, or ions) are arranged in a ...

X-ray crystallography
work done by
Rosalind Franklin Rosalind Elsie Franklin (25 July 192016 April 1958) was an English chemist A chemist (from Greek ''chēm(ía)'' alchemy; replacing ''chymist'' from Medieval Latin Medieval Latin was the form of Latin Latin (, or , ) is a classica ...

Rosalind Franklin
and
Maurice Wilkins Maurice Hugh Frederick Wilkins (15 December 1916 – 5 October 2004) was a New Zealand-born and whose research spanned multiple areas of physics and biophysics, contributing to the scientific understanding of , , and , and to the developme ...
. Watson and Crick described the structure of DNA and conjectured about the implications of this unique structure for possible mechanisms of DNA replication. J. D. Watson and F. H. C. Crick were awarded Nobel prize in 1962, along with Maurice Wilkens, for proposing a model of the structure of DNA. As time pass by, in 1964 K. A. Marcker and Frederick Sanger discovered a peculiar amioacyl-tRNA in E.coli , called N-formyl- methionyl – tRNA and explained that this molecule play a role in special mechanism of the chain elongation. He was awarded second Nobel prize for discovering complete sequence of 5,400 nucleotides of single stranded DNA of F ´ 174 bacteriophages. In 1961, it was demonstrated that when a
gene In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mecha ...

gene
encodes a
protein Proteins are large biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowdery Kendrew in 1958, for which they received a No ...

protein
, three sequential bases of a gene's
DNA Deoxyribonucleic acid (; DNA) is a molecule File:Pentacene on Ni(111) STM.jpg, A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A molecule is an electrically neutral gro ...

DNA
specify each successive amino acid of the protein. Thus the
genetic code The genetic code is the set of rules used by living cell (biology), cells to Translation (biology), translate information encoded within genetic material (DNA or Messenger RNA, mRNA sequences of nucleotide triplets, or codons) into proteins. Tran ...

genetic code
is a triplet code, where each triplet (called a codon) specifies a particular amino acid. Furthermore, it was shown that the codons do not overlap with each other in the DNA sequence encoding a protein, and that each sequence is read from a fixed starting point. During 1962-1964, through the use of conditional lethal mutants of a bacterial virus, fundamental advances were made in our understanding of the functions and interactions of the proteins employed in the machinery of
DNA replication In , DNA replication is the of producing two identical replicas of DNA from one original molecule. DNA replication occurs in all acting as the most essential part for . This is essential for cell division during growth and repair of damaged tis ...

DNA replication
,
DNA repair DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA Deoxyribonucleic acid (; DNA) is a molecule File:Pentacene on Ni(111) STM.jpg, A scanning tunneling microscopy image of pentacene mol ...

DNA repair
, DNA recombination, and in the assembly of molecular structures.


The F.Griffith experiment

In 1928, Fredrick Griffith, encountered a virulence property in pneumococcus bacteria, which was killing lab rats. According to Mendel, prevalent at that time, gene transfer could occur only from parent to daughter cells only. Griffith advanced another theory, stating that gene transfer occurring in member of same generation is known as horizontal gene transfer (HGT). This phenomenon is now referred to as genetic transformation. Griffith addressed the ''Streptococcus pneumoniae'' bacteria, which had are two different strains, one virulent and smooth and one avirulent and rough. The smooth strain had glistering appearance owing to the presence of a type of specific polysaccharide – a polymer of glucose and glucuronic acid capsule. Due to this polysaccharide layer of bacteria, a host's immune system cannot recognize the bacteria and it kills the host. The other, avirulent, rough strain lacks this polysaccharide capsule and has a dull, rough appearance. Presence or absence of capsule in the  strain, is known to be genetically determined. Smooth and rough strains occur in several different type such as S-I, S-II , S-III, etc. and R-I, R-II, R-III, etc. respectively. All this subtypes of S and R bacteria differ with each other in antigen type they produce.


Hershey and Chase experiment

Confirmation that DNA is the genetic material which is cause of infection came from Hershey and Chase experiment. They used ''E.coli'' and bacteriophage for the experiment. This experiment is also known as blender experiment, as kitchen blender was used as a major piece of apparatus. ''Alfred Hershey'' and ''Martha Chase'' demonstrated that the DNA injected by a phage particle into a bacterium contains all information required to synthesize progeny phage particles. They used radioactivity to tag the bacteriophage's protein coat with radioactive sulphur and DNA with radioactive phosphorus, into two different test tubes respectively. After mixing bacteriophage and ''E.coli'' into the test tube, the incubation period starts in which phage transforms the genetic material in the ''E.coli'' cells. Then the mixture is blended or agitated, which separates the phage from ''E.coli'' cells. The whole mixture is centrifuged and the pellet which contains ''E.coli'' cells was checked and the supernatant was discarded. The ''E.coli'' cells showed radioactive phosphorus, which indicated that the transformed material was DNA not the protein coat. The transformed DNA gets attached to the DNA of ''E.coli'' and radioactivity is only seen onto the bacteriophage's DNA. This mutated DNA can be passed to the next generation and the theory of Transduction came into existence. Transduction is a process in which the bacterial DNA carry the fragment of bacteriophages and pass it on the next generation. This is also a type of horizontal gene transfer.


Relationship to other biological sciences

The following list describes a viewpoint on the interdisciplinary relationships between molecular biology and other related fields. * ''Molecular biology'' is the study of the molecular underpinnings of the biological phenomena, focusing on molecular synthesis, modification, mechanisms and interactions. * ''Biochemistry'' is the study of the chemical substances and vital processes occurring in living
organism In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological me ...

organism
s.
Biochemist Biochemists are scientists who are trained in biochemistry Biochemistry or biological chemistry, is the study of chemical process In a scientific Science (from the Latin Latin (, or , ) is a classical language belonging to the Ital ...
s focus heavily on the role, function, and structure of
biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowdery Kendrew in 1958, for which they received a Nobel Prize in Chemistry, Nobel Prize i ...
s such as
protein Proteins are large biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowdery Kendrew in 1958, for which they received a No ...

protein
s,
lipid In and , a lipid is a macro that is soluble in solvents. are typically s used to dissolve other naturally occurring hydrocarbon lipid s that do not (or do not easily) dissolve in water, including s, es, s, fat-soluble s (such as vitamins A, ...
s,
carbohydrate is a disaccharide A disaccharide (also called a double sugar or ''biose'') is the sugar formed when two monosaccharides are joined by glycosidic linkage. Like monosaccharides, disaccharides are simple sugars soluble in water. Three common ex ...
s and
nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all Organism, known forms of life. They are composed of nucleotides, which are the monomers made of three components: a pentose, 5-carbon sugar, a phosphate group and a nitrogenous base. ...

nucleic acid
s. * ''Genetics'' is the study of how genetic differences affect organisms.
Genetics Genetics is a branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, ...

Genetics
attempts to predict how
mutation In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mechan ...
s, individual
gene In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mecha ...

gene
s and
genetic interactions File:Epistasis.png, Example of epistasis in coat colour genetics: If no pigments can be produced the other coat colour genes have no effect on the phenotype, no matter if they are dominant or if the individual is homozygous. Here the genotype "c ...

genetic interactions
can affect the expression of a
phenotype In genetics Genetics is a branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular inter ...

phenotype
While researchers practice techniques specific to molecular biology, it is common to combine these with methods from
genetics Genetics is a branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, ...

genetics
and
biochemistry Biochemistry or biological chemistry, is the study of es within and relating to living s. A sub-discipline of both and , biochemistry may be divided into three fields: , and . Over the last decades of the 20th century, biochemistry has beco ...

biochemistry
. Much of molecular biology is quantitative, and recently a significant amount of work has been done using computer science techniques such as
bioinformatics Bioinformatics () is an interdisciplinary field that develops methods and software tools for understanding biology, biological data, in particular when the data sets are large and complex. As an interdisciplinary field of science, bioinformat ...

bioinformatics
and
computational biology Computational biology involves the development and application of data-analytical and theoretical methods, and computational simulation techniques to the study of biological, , behavioural, and social systems. The field is broadly defined and incl ...

computational biology
.
Molecular genetics Molecular genetics is a sub-field of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the ...
, the study of gene structure and function, has been among the most prominent sub-fields of molecular biology since the early 2000s. Other branches of biology are informed by molecular biology, by either directly studying the interactions of molecules in their own right such as in
cell biology Cell biology (also cellular biology or cytology) is a branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, ...
and
developmental biology Developmental biology is the study of the process by which animal Animals (also called Metazoa) are multicellular A multicellular organism is an organism In biology, an organism () is any organic, life, living system that fu ...
, or indirectly, where molecular techniques are used to infer historical attributes of
population Population typically refers the number of people in a single area whether it be a city or town, region, country, or the world. Governments typically quantify the size of the resident population within their jurisdiction by a process called a ...

population
s or
species In biology, a species is the basic unit of biological classification, classification and a taxonomic rank of an organism, as well as a unit of biodiversity. A species is often defined as the largest group of organisms in which any two individu ...

species
, as in fields in
evolutionary biology Evolutionary biology is the subfield of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interacti ...
such as
population genetics Population genetics is a subfield of that deals with genetic differences within and between s, and is a part of . Studies in this branch of examine such phenomena as , , and . Population genetics was a vital ingredient in the of the . Its pri ...
and
phylogenetics In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mechanism ...

phylogenetics
. There is also a long tradition of studying
biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowdery Kendrew in 1958, for which they received a Nobel Prize in Chemistry, Nobel Prize i ...
s "from the ground up", or molecularly, in
biophysics uses protein domain dynamics on nanoscale Image:Protein translation.gif, 300px, A ribosome is a biological machine that utilizes nanoscale protein dynamics The nanoscopic scale (or nanoscale) usually refers to structures with a length scale ...
.


Techniques of molecular biology


Molecular cloning

Molecular cloning is used to isolate and then transfer a DNA sequence of interest into a plasmid vector. This recombinant DNA technology was first developed in the 1960's. In this technique, a
DNA Deoxyribonucleic acid (; DNA) is a molecule File:Pentacene on Ni(111) STM.jpg, A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A molecule is an electrically neutral gro ...

DNA
sequence coding for a protein of interest is
cloned Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, clo ...
using
polymerase chain reaction Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete copies or partial copies) of a specific DNA Deoxyribonucleic acid (; DNA) is a molecule File:Pentacene on Ni(111) STM. ...

polymerase chain reaction
(PCR), and/or
restriction enzyme A restriction enzyme, restriction endonuclease, or '' restrictase '' is an enzyme Enzymes () are protein Proteins are large s and s that comprise one or more long chains of . Proteins perform a vast array of functions within organism ...

restriction enzyme
s, into a
plasmid A plasmid is a small, extrachromosomal DNA Extrachromosomal DNA (abbreviated ecDNA) is any DNA that is found off the chromosome A chromosome is a long DNA molecule with part or all of the genetic material of an organism. Most eukaryo ...
(
expression vector An expression vector, otherwise known as an expression construct, is usually a plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They a ...
). The plasmid vector has 3 distinctive features: an origin of replication, a
multiple cloning siteA multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only onc ...
(MCS), and a selective marker (usually
antibiotic resistance Antimicrobial resistance (AMR or AR) occurs when microbe A microorganism, or microbe,, ''mikros'', "small") and ''organism'' from the el, ὀργανισμός, ''organismós'', "organism"). It is usually written as a single word but is some ...

antibiotic resistance
). Additionally, upstream of the MCS are the
promoter region In genetics, a promoter is a sequence of DNA to which proteins bind that initiate transcription (genetics), transcription of a single RNA from the DNA downstream of it. This RNA may encode a protein, or can have a function in and of itself, such ...
s and the transcription start site, which regulate the expression of cloned gene. This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by
transformation Transformation may refer to: Science and mathematics In biology and medicine * Metamorphosis, the biological process of changing physical form after birth or hatching * Malignant transformation, the process of cells becoming cancerous * Transf ...
via uptake of naked DNA,
conjugation Conjugation or conjugate may refer to: Linguistics * Grammatical conjugation, the modification of a verb from its basic form * Emotive conjugation or Russell's conjugation, the use of loaded language Mathematics * Complex conjugation, the change ...
via cell-cell contact or by via viral vector. Introducing DNA into
eukaryotic Eukaryotes () are organism In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interact ...

eukaryotic
cells, such as animal cells, by physical or chemical means is called
transfection Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic Eukaryotes () are organism In biology, an organism (from Ancient Greek, Greek: ὀργανισμός, ''organismos'') is any indi ...

transfection
. Several different transfection techniques are available, such as calcium phosphate transfection,
electroporation Electroporation, or electropermeabilization, is a microbiology Microbiology (from Greek Greek may refer to: Greece Anything of, from, or related to Greece Greece ( el, Ελλάδα, , ), officially the Hellenic Republic, is a country loca ...
,
microinjection Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic The microscopic scale (from , ''mikrós'', "small" and σκοπέω, ''skopéō'' "look") is the scale of objects and events smaller than those that c ...
and liposome transfection. The plasmid may be integrated into the
genome In the fields of molecular biology and genetics, a genome is all genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The genome includes both the genes (the coding regions) and the noncodin ...

genome
, resulting in a stable transfection, or may remain independent of the genome, called transient transfection. DNA coding for a protein of interest is now inside a cell, and the
protein Proteins are large biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowdery Kendrew in 1958, for which they received a No ...

protein
can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help 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 under a variety of situations, the protein may be crystallized so its
tertiary structure Biomolecular structure is the intricate folded, three-dimensional shape that is formed by a molecule A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A molecule is an elect ...

tertiary structure
can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.


Polymerase chain reaction

Polymerase chain reaction Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete copies or partial copies) of a specific DNA Deoxyribonucleic acid (; DNA) is a molecule File:Pentacene on Ni(111) STM. ...

Polymerase chain reaction
(PCR) is an extremely versatile technique for copying DNA. In brief, PCR allows a specific
DNA sequence DNA sequencing is the process of determining the nucleic acid sequence A nucleic acid sequence is a succession of bases signified by a series of a set of five different letters that indicate the order of nucleotides Nucleotides are organic ...

DNA sequence
to be copied or modified in predetermined ways. The reaction is extremely powerful and under perfect conditions could amplify one DNA molecule to become 1.07 billion molecules in less than two hours. PCR has many applications, including the study of gene expression, the detection of pathogenic microorganisms, the detection of genetic mutations, and the introduction of mutations to DNA. The PCR technique can be used to introduce restriction enzyme sites to ends of DNA molecules, or to mutate particular bases of DNA, the latter is a method referred to as
site-directed mutagenesisSite-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene In biology, a gene (from ''genos'' "...Wilhelm Johannsen coined the word gene to describe the Mendeli ...
. PCR can also be used to determine whether a particular DNA fragment is found in a
cDNA library A cDNA library is a combination of cloned cDNA ( complementary DNA) fragments inserted into a collection of host cells, which constitute some portion of the transcriptome of the organism and are stored as a "library A library is a curated co ...
. PCR has many variations, like reverse transcription PCR (
RT-PCR Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chai ...
) for amplification of RNA, and, more recently,
quantitative PCR A real-time polymerase chain reaction (real-time PCR), also known as quantitative polymerase chain reaction (qPCR), is a laboratory technique of molecular biology based on the polymerase chain reaction#REDIRECT Polymerase chain reaction {{Re ...
which allow for quantitative measurement of DNA or RNA molecules.


Gel electrophoresis

Gel electrophoresis File:Gel Electrophoresis in DNA Fingerprinting.svg, 200px, Gel electrophoresis is a process where an electric current is applied to DNA samples creating fragments that can be used for comparison between DNA samples. 1) DNA is extracted.2) Isola ...

Gel electrophoresis
is a technique which separates molecules by their size using an agarose or polyacrylamide gel. This technique is one of the principal tools of molecular biology. The basic principle is that DNA fragments can be separated by applying an electric current across the gel - because the DNA backbone contains negatively charged phosphate groups, the DNA will migrate through the agarose gel towards the positive end of the current. Proteins can also be separated on the basis of size using an
SDS-PAGE SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis), is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate protein Proteins are large biomolecules or macrom ...

SDS-PAGE
gel, or on the basis of size and their
electric charge Electric charge is the physical property of matter that causes it to experience a force when placed in an electromagnetic field. Electric charge can be ''positive'' or ''negative'' (commonly carried by protons and electrons respectively). Like c ...
by using what is known as a 2D gel electrophoresis.


The Bradford Assay

The Bradford Assay is a molecular biology technique which enables the fast, accurate quantitation of protein molecules utilizing the unique properties of a dye called
Coomassie Brilliant Blue Coomassie Brilliant Blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining specimen, sandwiched between a glass microscope slide. Staining is a technique ...
G-250. Coomassie Blue undergoes a visible color shift from reddish-brown to bright blue upon binding to protein. In its unstable, cationic state, Coomassie Blue has a background wavelength of 465 nm and gives off a reddish-brown color. When Coomassie Blue binds to protein in an acidic solution, the background wavelength shifts to 595 nm and the dye gives off a bright blue color. Proteins in the assay bind Coomassie blue in about 2 minutes, and the protein-dye complex is stable for about an hour, although it's recommended that absorbance readings are taken within 5 to 20 minutes of reaction initiation. The concentration of protein in the Bradford assay can then be measured using a visible light , and therefore does not require extensive equipment. This method was developed in 1975 by Marion M. Bradford, and has enabled significantly faster, more accurate protein quantitation compared to previous methods: the Lowry procedure and the biuret assay. Unlike the previous methods, the Bradford assay is not susceptible to interference by several non-protein molecules, including ethanol, sodium chloride, and magnesium chloride.  However, it is susceptible to influence by strong alkaline buffering agents, such as
sodium dodecyl sulfate Sodium dodecyl sulfate (SDS) or sodium lauryl sulfate (SLS), sometimes written sodium laurilsulfate, is a synthetic organic compound , CH4; is among the simplest organic compounds. In chemistry, organic compounds are generally any chemical compou ...

sodium dodecyl sulfate
(SDS).


Macromolecule blotting and probing

The terms ''northern'', ''western'' and ''eastern'' blotting are derived from what initially was a molecular biology joke that played on the term ''
Southern blot Southern blot membrane after hybridization and rinsing. A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis Gel electrophore ...
ting'', after the technique described by
Edwin Southern Sir Edwin Mellor Southern (born 7 June 1938) is an English Lasker Award The Lasker Awards have been awarded annually since 1945 to living persons who have made major contributions to medical science or who have performed public service on behal ...
for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as the ''northern blot'', actually didn't use the term.


Southern blotting

Named after its inventor, biologist
Edwin Southern Sir Edwin Mellor Southern (born 7 June 1938) is an English Lasker Award The Lasker Awards have been awarded annually since 1945 to living persons who have made major contributions to medical science or who have performed public service on behal ...
, the
Southern blot Southern blot membrane after hybridization and rinsing. A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis Gel electrophore ...
is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after
restriction enzyme A restriction enzyme, restriction endonuclease, or '' restrictase '' is an enzyme Enzymes () are protein Proteins are large s and s that comprise one or more long chains of . Proteins perform a vast array of functions within organism ...

restriction enzyme
(restriction endonuclease) digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via
capillary action Capillary action (sometimes capillarity, capillary motion, capillary effect, or wicking) is the ability of a liquid A liquid is a nearly incompressible In fluid mechanics Fluid mechanics is the branch of physics concerned with the ...
. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as
PCR Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete copies or partial copies) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a pa ...

PCR
, to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring
transgene A transgene is a gene In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiol ...
copy number in
transgenic mice A genetically modified mouse or genetically engineered mouse model (GEMM) is a mouse A mouse, plural mice, is a small rodent Rodents (from Latin Latin (, or , ) is a classical language belonging to the Italic languages, Italic branc ...
or in the engineering of
gene knockout A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemi ...
embryonic stem cell lines.


Northern blotting

The northern blot is used to study the presence of specific RNA molecules as relative comparison among a set of different samples of RNA. It is essentially a combination of denaturing gel, denaturing RNA gel electrophoresis, and a blot (biology), blot. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complementarity (molecular biology), complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples, assuming that no post-transcriptional regulation occurs and that the levels of mRNA reflect proportional levels of the corresponding protein being produced. It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues.


Western blotting

A western blot is a technique by which specific proteins can be detected from a mixture of proteins. Western blots can be used to determine the size of isolated proteins, as well as to quantify their expression. In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as
SDS-PAGE SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis), is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate protein Proteins are large biomolecules or macrom ...

SDS-PAGE
. The proteins in the gel are then transferred to a polyvinylidene fluoride (PVDF), nitrocellulose, nylon, or other support membrane. This membrane can then be probed with solutions of antibody, antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with enzymes. When a chemiluminescent Substrate (biochemistry), substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis. Analogous methods to western blotting can be used to directly stain specific proteins in live cell (biology), cells or biological tissue, tissue sections.


Eastern blotting

The eastern blotting technique is used to detect post-translational modification of proteins. Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications using specific substrates.


Microarrays

A DNA microarray is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragments. Arrays make it possible to put down large quantities of very small (100 micrometre diameter) spots on a single slide. Each spot has a DNA fragment molecule that is complementary to a single
DNA sequence DNA sequencing is the process of determining the nucleic acid sequence A nucleic acid sequence is a succession of bases signified by a series of a set of five different letters that indicate the order of nucleotides Nucleotides are organic ...

DNA sequence
. A variation of this technique allows the gene expression of an organism at a particular stage in development to be qualified (expression profiling). In this technique the RNA in a tissue is isolated and converted to labeled complementary DNA (cDNA). This cDNA is then hybridized to the fragments on the array and visualization of the hybridization can be done. Since multiple arrays can be made with exactly the same position of fragments, they are particularly useful for comparing the gene expression of two different tissues, such as a healthy and cancerous tissue. Also, one can measure what genes are expressed and how that expression changes with time or with other factors. There are many different ways to fabricate microarrays; the most common are silicon chips, microscope slides with spots of ~100 micrometre diameter, custom arrays, and arrays with larger spots on porous membranes (macroarrays). There can be anywhere from 100 spots to more than 10,000 on a given array. Arrays can also be made with molecules other than DNA.


Allele-specific oligonucleotide

Allele-specific oligonucleotide (ASO) is a technique that allows detection of single base mutations without the need for PCR or gel electrophoresis. Short (20–25 nucleotides in length), labeled probes are exposed to the non-fragmented target DNA, hybridization occurs with high specificity due to the short length of the probes and even a single base change will hinder hybridization. The target DNA is then washed and the labeled probes that didn't hybridize are removed. The target DNA is then analyzed for the presence of the probe via radioactivity or fluorescence. In this experiment, as in most molecular biology techniques, a control must be used to ensure successful experimentation. In molecular biology, procedures and technologies are continually being developed and older technologies abandoned. For example, before the advent of DNA gel electrophoresis (agarose gel electrophoresis, agarose or SDS-PAGE, polyacrylamide), the size of DNA molecules was typically determined by rate sedimentation in sucrose gradient centrifugation, sucrose gradients, a slow and labor-intensive technique requiring expensive instrumentation; prior to sucrose gradients, viscometry was used. Aside from their historical interest, it is often worth knowing about older technology, as it is occasionally useful to solve another new problem for which the newer technique is inappropriate.


See also


References


Further reading

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External links

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