Molecular biology is the branch of
biology
Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditary i ...
that seeks to understand the
molecular basis of biological activity in and between
cells, including
biomolecular
A biomolecule or biological molecule is a loosely used term for molecules present in organisms that are essential to one or more typically biological processes, such as cell division, morphogenesis, or development. Biomolecules include large ...
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. In 1953 Francis Crick, James Watson, Rosalind Franklin, and colleagues, working at Medical Research Council unit, Cavendish laboratory, Cambridge (now the
MRC Laboratory of Molecular Biology
The Medical Research Council (MRC) Laboratory of Molecular Biology (LMB) is a research institute in Cambridge, England, involved in the revolution in molecular biology which occurred in the 1950–60s. Since then it has remained a major medical r ...
), made a
double helix
A double is a look-alike or doppelgänger; one person or being that resembles another.
Double, The Double or Dubble may also refer to:
Film and television
* Double (filmmaking), someone who substitutes for the credited actor of a character
* ...
model of
DNA which changed the entire research scenario. They proposed the DNA structure based on previous research done by Rosalind Franklin and Maurice Wilkins. This research then 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 a collection of techniques developed since the field's genesis which have enabled scientists to learn about molecular processes.
In this way it has both complemented and improved
biochemistry
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...
and
genetics
Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar wor ...
as methods (of understanding
nature
Nature, in the broadest sense, is the physics, physical world or universe. "Nature" can refer to the phenomenon, phenomena of the physical world, and also to life in general. The study of nature is a large, if not the only, part of science. ...
) that began before its advent. One notable technique which has revolutionized the field is the
polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(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.
The
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, all of which can be used to efficiently target new
drug
A drug is any chemical substance that causes a change in an organism's physiology or psychology when consumed. Drugs are typically distinguished from food and substances that provide nutritional support. Consumption of drugs can be via insuffla ...
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 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 human DN ...
whereas the use of molecular biology or
molecular cell biology in medicine is now referred to as
molecular medicine
Molecular medicine is a broad field, where physical, chemical, biological, bioinformatics and medical techniques are used to describe molecular structures and mechanisms, identify fundamental molecular and genetic errors of disease, and to develop ...
.
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.
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 or sexual reproduction, the offspring cells or organisms acquire the genetic informa ...
and the structure of a
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
.
Gregor Mendel
Gregor Johann Mendel, OSA (; cs, Řehoř Jan Mendel; 20 July 1822 – 6 January 1884) was a biologist, meteorologist, mathematician, Augustinian friar and abbot of St. Thomas' Abbey in Brünn (''Brno''), Margraviate of Moravia. Mendel was ...
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 (also known as Mendelism) is a type of biological inheritance following the principles originally proposed by Gregor Mendel in 1865 and 1866, re-discovered in 1900 by Hugo de Vries and Carl Correns, and later popularized ...
, which states that diploid individuals with two
allele
An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is a variation of the same sequence of nucleotides at the same place on a long DNA molecule, as described in leading textbooks on genetics and evolution.
::"The chro ...
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 (also known as Mendelism) is a type of biological inheritance following the principles originally proposed by Gregor Mendel in 1865 and 1866, re-discovered in 1900 by Hugo de Vries and Carl Correns, and later populari ...
.
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 to isolate nucleic acid in 1869. He also identified protamine and made a number of other discoveries.
Miescher had i ...
, 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 a Russian born American biochemist who studied the structure and function of nucleic acids. He characterized the different forms of nucleic acid, DNA from RNA, and fo ...
, 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, writer, Bucovinian Jew who emigrated to the United States during the Nazi era, and professor of biochemistry at Columbia University medical schoo ...
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 and
Francis Crick published the double helical structure of DNA,
using the
X-ray crystallography
X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
work done by
Rosalind Franklin and
Maurice Wilkins. 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.
In 1961, it was demonstrated that when a
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
encodes a
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
, three sequential bases of a gene's DNA specify each successive amino acid of the protein. Thus the
genetic code
The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...
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 molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
,
DNA repair
DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA dam ...
,
DNA recombination, and in the assembly of molecular structures.
The F.Griffith experiment
![01 dna](https://upload.wikimedia.org/wikipedia/commons/2/2c/01_dna.jpg)
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 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
![Hershey Chase experiment](https://upload.wikimedia.org/wikipedia/commons/3/32/Hershey_Chase_experiment.png)
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.
Modern molecular biology
In the early 2020s, molecular biology entered a golden age defined by both vertical and horizontal technical development. Vertically, novel technologies are allowing for real-time monitoring of biological processes at the atomic level. Molecular biologists today have access to increasingly affordable sequencing data at increasingly higher depths, facilitating the development of novel genetic manipulation methods in new non-model organisms. Likewise, synthetic molecular biologists will drive the industrial production of small and macro molecules through the introduction of exogenous metabolic pathways in various prokaryotic and eukaryotic cell lines.
Horizontally, sequencing data is becoming more affordable and used in many different scientific fields. This will drive the development of industries in developing nations and increase accessibility to individual researchers. Likewise, CRISPR-Cas9 gene editing experiments can now be conceived and implemented by individuals for under $10,000 in novel organisms, which will drive the development of industrial and medical applications
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, an organism () is any living system that functions as an individual entity. All organisms are composed of cells (cell theory). Organisms are classified by taxonomy into groups such as multicellular animals, plants, and ...
s.
Biochemists focus heavily on the role, function, and structure of
biomolecules such as
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s,
lipid
Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...
s,
carbohydrate
In organic chemistry, a carbohydrate () is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula (where ''m'' may or ma ...
s and
nucleic acid
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
s.
* ''Genetics'' is the study of how genetic differences affect organisms.
Genetics
Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar wor ...
attempts to predict how
mutation
In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mi ...
s, individual
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
s and
genetic interactions
Epistasis is a phenomenon in genetics in which the effect of a gene mutation is dependent on the presence or absence of mutations in one or more other genes, respectively termed modifier genes. In other words, the effect of the mutation is dep ...
can affect the expression of a
phenotype
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
While researchers practice techniques specific to molecular biology, it is common to combine these with methods from
genetics
Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar wor ...
and
biochemistry
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...
. 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 biological data, in particular when the data sets are large and complex. As an interdisciplinary field of science, bioinformatics combi ...
and
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 that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living and ...
and
developmental biology, or indirectly, where molecular techniques are used to infer historical attributes of
population
Population typically refers to the number of people in a single area, whether it be a city or town, region, country, continent, or the world. Governments typically quantify the size of the resident population within their jurisdiction using a ...
s or
species
In biology, a species is the basic unit of 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 individuals of the appropriate s ...
, as in fields in
evolutionary biology
Evolutionary biology is the subfield of biology that studies the evolutionary processes (natural selection, common descent, speciation) that produced the diversity of life on Earth. It is also defined as the study of the history of life fo ...
such as
population genetics
Population genetics is a subfield of genetics that deals with genetic differences within and between populations, and is a part of evolutionary biology. Studies in this branch of biology examine such phenomena as adaptation, speciation, and pop ...
and
phylogenetics
In biology, phylogenetics (; from Greek language, Greek wikt:φυλή, φυλή/wikt:φῦλον, φῦλον [] "tribe, clan, race", and wikt:γενετικός, γενετικός [] "origin, source, birth") is the study of the evolutionary his ...
. There is also a long tradition of studying
biomolecules "from the ground up", or molecularly, in
biophysics
Biophysics is an interdisciplinary science that applies approaches and methods traditionally used in physics to study biological phenomena. Biophysics covers all scales of biological organization, from molecular to organismic and populations. ...
.
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 1960s. In this technique, a
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, ...
using
polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(PCR), and/or
restriction enzyme
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
s, into 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 are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
(
expression vector
An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for ...
). The plasmid vector usually has at least 3 distinctive features: an origin of replication, a
multiple cloning site A 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 ...
(MCS), and a selective marker (usually
antibiotic resistance
Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistance. ...
). Additionally, upstream of the MCS are the
promoter region
In genetics, a promoter is a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. The RNA transcript may encode a protein (mRNA), or can have a function in and of ...
s and the
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
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
* Tran ...
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 chang ...
via cell-cell contact or by
transduction via viral vector. Introducing DNA into
eukaryotic
Eukaryotes () are organisms whose Cell (biology), cells have a cell nucleus, nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the ...
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 cells. It may also refer to other methods and cell types, although other terms are often preferred: " transformation" is typically used to des ...
. Several different transfection techniques are available, such as calcium phosphate transfection,
electroporation
Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introd ...
,
microinjection
Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical pro ...
and
liposome transfection. The plasmid may be integrated into the
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
, resulting in a stable transfection, or may remain independent of the genome and expressed temporarily, called a transient transfection.
DNA coding for a protein of interest is now inside a cell, and the
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
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
Protein tertiary structure is the three dimensional shape of a protein. The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains. Amino acid side chains may i ...
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
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(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 – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
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 mutagenesis
Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesi ...
. PCR can also be used to determine whether a particular DNA fragment is found in a
cDNA library. 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 ch ...
) for amplification of RNA, and, more recently,
quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
which allow for quantitative measurement of DNA or RNA molecules.
Gel electrophoresis
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 sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...
gel, or on the basis of size and their
electric charge
Electric charge is the physical property of matter that causes charged matter to experience a force when placed in an electromagnetic field. Electric charge can be ''positive'' or ''negative'' (commonly carried by protons and electrons respe ...
by using what is known as a
2D gel electrophoresis.
The Bradford Assay
The Bradford Assay
The Bradford protein assay was developed by Marion M. Bradford in 1976. It is a quick and accurate spectroscopic analytical procedure used to measure the concentration of protein in a solution. The reaction is dependent on the amino acid composi ...
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 proteins in analytical biochemistry. Coomassie brilliant blue G-250 differs from ...
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
spectrophotometer, 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 an organic compound with the formula . It is an anionic surfactant used in many cleaning and hygiene products. This compound is the sodium salt ...
(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 blotting'', after the technique described by
Edwin Southern
Sir Edwin Mellor Southern (born 7 June 1938) is an English Lasker Award-winning molecular biologist, Emeritus Professor of Biochemistry at the University of Oxford and a fellow of Trinity College, Oxford. He is most widely known for the inventio ...
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-winning molecular biologist, Emeritus Professor of Biochemistry at the University of Oxford and a fellow of Trinity College, Oxford. He is most widely known for the inventio ...
, the
Southern blot 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, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
(restriction endonuclease) digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via
capillary action
Capillary action (sometimes called capillarity, capillary motion, capillary rise, capillary effect, or wicking) is the process of a liquid flowing in a narrow space without the assistance of, or even in opposition to, any external forces li ...
. 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, 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 that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change th ...
copy number in
transgenic mice
A genetically modified mouse or genetically engineered mouse model (GEMM) is a mouse (''Mus musculus'') that has had its genome altered through the use of genetic engineering techniques. Genetically modified mice are commonly used for research or ...
or in the engineering of
gene knockout embryonic stem cell lines.
Northern blotting
![Northern blot diagram](https://upload.wikimedia.org/wikipedia/commons/4/4d/Northern_blot_diagram.png)
The
northern blot
The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample ...
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 RNA gel electrophoresis, and a
blot
Blot may refer to:
Surname
* Guillaume Blot (born 1985), French racing cyclist
* Harold W. Blot (born 1938), served as United States Deputy Chief of Staff for Aviation
* Jean-François Joseph Blot (1781–1857), French soldier and politician
*Yv ...
. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled
complement
A complement is something that completes something else.
Complement may refer specifically to:
The arts
* Complement (music), an interval that, when added to another, spans an octave
** Aggregate complementation, the separation of pitch-clas ...
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 blot
The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...
ting, 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 sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...
. The proteins in the gel are then transferred to a
polyvinylidene fluoride
Polyvinylidene fluoride or polyvinylidene difluoride (PVDF) is a highly non-reactive thermoplastic fluoropolymer produced by the polymerization of vinylidene difluoride.
PVDF is a specialty plastic used in applications requiring the highest pur ...
(PVDF), nitrocellulose, nylon, or other support membrane. This membrane can then be probed with solutions of
antibodies
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
. 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
Chemiluminescence (also chemoluminescence) is the emission of light (luminescence) as the result of a chemical reaction. There may also be limited emission of heat. Given reactants A and B, with an excited intermediate ◊,
: + -> lozenge - ...
substrate is exposed to the
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
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
cells or
tissue sections.
Eastern blotting
The
eastern blot
The eastern blot, or eastern blotting, is a biochemical technique used to analyze protein post-translational modifications including the addition of lipids, phosphates, and glycoconjugates. It is most often used to detect carbohydrate epitopes. Thu ...
ting technique is used to detect
post-translational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribos ...
of proteins. Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications using specific substrates.
Microarrays
![NA hybrid](https://upload.wikimedia.org/wikipedia/commons/a/a8/NA_hybrid.svg)
A
DNA microarray is a collection of spots attached to a solid support such as a
microscope slide
A microscope slide is a thin flat piece of glass, typically 75 by 26 mm (3 by 1 inches) and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is mounted (secured) on the slide, and then b ...
where each spot contains one or more single-stranded DNA
oligonucleotide
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids ...
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 – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
. A variation of this technique allows the
gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
of an organism at a particular stage in development to be qualified (
expression profiling
In the field of molecular biology, gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between c ...
). In this technique the RNA in a tissue is isolated and converted to labeled
complementary DNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spec ...
(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 An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler Dot bl ...
(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
Agarose is a heteropolysaccharide, generally extracted from certain red seaweed. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is ...
or
polyacrylamide
Polyacrylamide (abbreviated as PAM) is a polymer with the formula (-CH2CHCONH2-). It has a linear-chain structure. PAM is highly water-absorbent, forming a soft gel when hydrated. In 2008, an estimated 750,000,000 kg were produced, mainly f ...
), the size of DNA molecules was typically determined by rate
sedimentation
Sedimentation is the deposition of sediments. It takes place when particles in suspension settle out of the fluid in which they are entrained and come to rest against a barrier. This is due to their motion through the fluid in response to the ...
in
sucrose gradients, a slow and labor-intensive technique requiring expensive instrumentation; prior to sucrose gradients,
viscometry
A viscometer (also called viscosimeter) is an instrument used to measure the viscosity of a fluid. For liquids with viscosities which vary with flow conditions, an instrument called a rheometer is used. Thus, a rheometer can be considered as a spe ...
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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