Editing Molecular biology

{{Short description|Branch of biology that studies biological systems at the molecular level}}
{{Redirect|Biochemical genetics|the scientific journal|Biochemical Genetics (journal){{!}}''Biochemical Genetics'' (journal)}}
{{redirect|Molecular microbiology|the scientific journal|Molecular Microbiology (journal){{!}}''Molecular Microbiology'' (journal)}}

{{TopicTOC-Biology}}'''Molecular biology''' {{IPAc-en|m|ə|ˈ|l|ɛ|k|j|ʊ|l|ər}} is a branch of [[biology]] that seeks to understand the [[molecule|molecular]] basis of biological activity in and between [[Cell (biology)|cells]], including [[biomolecule|biomolecular]] synthesis, modification, mechanisms, and interactions.<ref name="cell2">{{cite book|last1=Alberts|first1=Bruce|url=https://books.google.com/books?id=jK6UBQAAQBAJ|title=Molecular Biology of the Cell, Sixth Edition|last2=Johnson|first2=Alexander|last3=Lewis|first3=Julian|last4=Morgan|first4=David|last5=Raff|first5=Martin|last6=Roberts|first6=Keith|last7=Walter|first7=Peter|date=2014|publisher=Garland Science|isbn=978-1-317-56375-4|pages=1–10|name-list-style=vanc}}</ref><ref name="Gannon-2002">{{cite journal | vauthors = Gannon F | title = Molecular biology--what's in a name? | journal = EMBO Reports | volume = 3 | issue = 2 | pages = 101 | date = February 2002 | pmid = 11839687 | pmc = 1083977 | doi = 10.1093/embo-reports/kvf039 }}</ref><ref>{{Cite web|title=Molecular biology – Latest research and news {{!}} Nature|url=https://www.nature.com/subjects/molecular-biology|access-date=2021-11-07|website=nature.com|language=en}}</ref>

Though cells and other microscopic structures had been observed in living organisms as early as the 18th century, a detailed understanding of the mechanisms and interactions governing their behavior did not emerge until the 20th century, when technologies used in physics and chemistry had advanced sufficiently to permit their application in the biological sciences. The term 'molecular biology' was first used in 1945 by the English physicist [[William Astbury]], who described it as an approach focused on discerning the underpinnings of biological phenomena—i.e. uncovering the physical and chemical structures and properties of biological molecules, as well as their interactions with other molecules and how these interactions explain observations of so-called classical biology, which instead studies biological processes at larger scales and higher levels of organization.<ref name="Astbury-1961">{{Cite journal|last=Astbury|first=W. T.|date=June 1961|title=Molecular Biology or Ultrastructural Biology ?|journal=Nature|language=en|volume=190|issue=4781|pages=1124|doi=10.1038/1901124a0|pmid=13684868|bibcode=1961Natur.190.1124A|s2cid=4172248|issn=1476-4687|doi-access=free}}</ref> In 1953, [[Francis Crick]], [[James Watson]], [[Rosalind Franklin]], and their colleagues at the [[MRC Laboratory of Molecular Biology|Medical Research Council Unit, Cavendish Laboratory]], were the first to describe the [[double helix]] model for the chemical structure of [[deoxyribonucleic acid]] (DNA), which is often considered a landmark event for the nascent field because it provided a physico-chemical basis by which to understand the previously nebulous idea of nucleic acids as the primary substance of biological inheritance. They proposed this structure based on previous research done by Franklin, which was conveyed to them by [[Maurice Wilkins]] and [[Max Perutz]].<ref name="Scitable-franklin">{{Cite web|title=Rosalind Franklin: A Crucial Contribution|url=https://www.nature.com/scitable/topicpage/rosalind-franklin-a-crucial-contribution-6538012/|website=nature.com|language=en}}</ref> Their work led to the discovery of DNA in other microorganisms, plants, and animals.<ref name="Verma-2004">{{Cite book |last=Verma |first= P. S. |title=Cell biology, genetics, molecular biology, evolution and ecology |date=2004 |publisher=S Chand and Company |isbn=81-219-2442-1 |oclc=1045495545 }}{{pn|date=June 2024}}</ref>

The field of molecular biology includes techniques which enable scientists to learn about molecular processes.<ref name="Morange-2016">{{cite book |doi=10.1002/9780470015902.a0003079.pub3 |chapter=History of Molecular Biology |title=Encyclopedia of Life Sciences |date=2016 |last1=Morange |first1=Michel |pages=1–8 |isbn=978-0-470-01617-6 }}</ref> These techniques are used to efficiently target new drugs, diagnose disease, and better understand cell physiology.<ref>{{Cite journal|last1=Bello|first1=Elizabeth A.|last2=Schwinn|first2=Debra A.|date=1996-12-01|title=Molecular Biology and Medicine: A Primer for the Clinician|journal=Anesthesiology|volume=85|issue=6|pages=1462–1478|doi=10.1097/00000542-199612000-00029|pmid=8968195|s2cid=29581630|issn=0003-3022|doi-access=free}}</ref> Some clinical research and medical therapies arising from molecular biology are covered under [[gene therapy]], whereas the use of molecular biology or [[molecular cell biology]] in medicine is now referred to as [[molecular medicine]].{{cn|date=September 2024}}

==History of molecular biology==
{{main|History of molecular biology}}
[[File:Hendiduras mayor menor-eo.png|thumb|Angle description in DNA structure]]
[[File:DNA Structure+Key+Labelled.png|thumb|Diagrammatic representation of Watson and Crick's DNA structure]]
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.<ref>{{Cite journal |last=Bynum |first=William |date=February 1999 |title=A History of Molecular Biology |url=https://www.nature.com/articles/nm0299_140 |journal=Nature Medicine |language=en |volume=5 |issue=2 |pages=140 |doi=10.1038/5498 |issn=1546-170X}}</ref><ref>{{Cite book|author=Morange, Michel |title=A history of biology|date=June 2021|publisher=Princeton University Press |isbn=978-0-691-18878-2|oclc=1184123419}}{{pn|date=June 2024}}</ref> Advances in molecular biology have been closely related to the development of new technologies and their optimization.<ref>{{Cite journal|last=Fields|first=Stanley|date=2001-08-28|title=The interplay of biology and technology|journal=Proceedings of the National Academy of Sciences|language=en|volume=98|issue=18|pages=10051–10054|doi=10.1073/pnas.191380098|issn=0027-8424|pmid=11517346|pmc=56913|doi-access=free}}</ref> 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.{{cn|date=May 2023}}

The field of genetics arose from attempts to understand the set of rules underlying [[reproduction]] and [[heredity]], and the nature of the hypothetical units of heredity known as [[gene]]s. [[Gregor Mendel]] pioneered this work in 1866, when he first described the laws of inheritance he observed in his studies of mating crosses in pea plants.<ref>{{cite journal |last1=Ellis |first1=T.H. Noel |last2=Hofer |first2=Julie M.I. |last3=Timmerman-Vaughan |first3=Gail M. |last4=Coyne |first4=Clarice J. |last5=Hellens |first5=Roger P. |title=Mendel, 150 years on |journal=Trends in Plant Science |date=November 2011 |volume=16 |issue=11 |pages=590–596 |doi=10.1016/j.tplants.2011.06.006 |pmid=21775188 |bibcode=2011TPS....16..590E }}</ref> One such law of genetic inheritance is the [[Law of Segregation|law of segregation]], which states that diploid individuals with two [[allele]]s for a particular gene will pass one of these alleles to their offspring.<ref>{{Cite web|date=2018-07-12|title=12.3C: Mendel's Law of Segregation|url=https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_General_Biology_(Boundless)/12%3A_Mendel%27s_Experiments_and_Heredity/12.3%3A_Laws_of_Inheritance/12.3C%3A_Mendels_Law_of_Segregation|access-date=2021-11-18|website=Biology LibreTexts|language=en}}</ref> Because of his critical work, the study of genetic inheritance is commonly referred to as [[Mendelian inheritance|Mendelian genetics]].<ref>{{Cite web|title=Mendelian Inheritance|url=https://www.genome.gov/genetics-glossary/Mendelian-Inheritance|access-date=2021-11-18|website=Genome.gov|language=en}}</ref>

A major milestone in molecular biology was the discovery of the structure of [[DNA]]. This work began in 1869 by [[Friedrich Miescher]], a Swiss biochemist who first proposed a structure called ''nuclein'', which we now know to be (deoxyribonucleic acid), or DNA.<ref name="Pray-2008">{{cite journal |last=Pray |first=L |date=2008 |title=Discovery of DNA structure and function: Watson and Crick |journal=Nature Education |volume=1 |issue=1 |page=100 |url=http://www.nature.com/scitable/topicpage/discovery-of-dna-structure-and-function-watson-397|access-date=2024-06-21}}</ref> He discovered this unique substance by studying the components of pus-filled bandages, and noting the unique properties of the "phosphorus-containing substances".<ref>{{Cite book|last=George.|first=Wolf |title=Friedrich Miescher: the man who discovered DNA|date=2003|oclc=907773747}}{{pn|date=June 2024}}</ref> Another notable contributor to the DNA model was [[Phoebus Levene]], who proposed the "polynucleotide model" of DNA in 1919 as a result of his biochemical experiments on yeast.<ref>{{Cite journal|last=Levene|first=P.A.|title=Structure of Yeast Nucleic Acid|date=1919|journal=Journal of Biological Chemistry|volume=43|issue=2|pages=379–382|doi=10.1016/s0021-9258(18)86289-5|issn=0021-9258|doi-access=free}}</ref> In 1950, [[Erwin Chargaff]] expanded on the work of Levene and elucidated a few critical properties of nucleic acids: first, the sequence of nucleic acids varies across species.<ref>{{cite journal |last1=Chargaff |first1=Erwin |title=Chemical specificity of nucleic acids and mechanism of their enzymatic degradation |journal=Experientia |date=June 1950 |volume=6 |issue=6 |pages=201–209 |doi=10.1007/bf02173653 |pmid=15421335 |s2cid=2522535 }}</ref> Second, the total concentration of purines (adenine and guanine) is always equal to the total concentration of pyrimidines (cysteine and thymine).<ref name="Pray-2008"/> This is now known as Chargaff's rule. In 1953, [[James Watson]] and [[Francis Crick]] published the double helical structure of DNA,<ref name="Watson-1953">{{Cite journal|last1=Watson|first1=J. D.|last2=Crick|first2=F. H. C.|authorlink1=James Watson|authorlink2=Francis Crick|date=April 1953|title=Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid|url=https://www.nature.com/articles/171737a0|journal=Nature|language=en|volume=171|issue=4356|pages=737–738|doi=10.1038/171737a0|pmid=13054692|bibcode=1953Natur.171..737W|s2cid=4253007|issn=1476-4687}}</ref> based on the [[X-ray crystallography]] work done by [[Rosalind Franklin]] which was conveyed to them by [[Maurice Wilkins]] and [[Max Perutz]].<ref name="Scitable-franklin" /> Watson and Crick described the structure of DNA and conjectured about the implications of this unique structure for possible mechanisms of DNA replication.<ref name="Watson-1953" /> Watson and Crick were awarded the [[Nobel Prize in Physiology or Medicine]] in 1962, along with Wilkins, for proposing a model of the structure of DNA.<ref name="Verma-2004" />

In 1961, it was demonstrated that when a [[gene]] encodes a [[protein]], three sequential bases of a gene's DNA specify each successive amino acid of the protein.<ref>{{cite journal | last1=Crick | first1=F. H. C. | last2=Barnett | first2=Leslie | last3=Brenner | first3=S. | last4=Watts-Tobin | first4=R. J. | title=General Nature of the Genetic Code for Proteins | journal=Nature | publisher=Springer Science and Business Media LLC | volume=192 | issue=4809 | year=1961 | issn=0028-0836 | doi=10.1038/1921227a0 | pages=1227–1232|bibcode=1961Natur.192.1227C|pmid=13882203|s2cid=4276146}}</ref> Thus the [[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,<ref>{{cite journal | last1=Epstein | first1=R. H. | last2=Bolle | first2=A. | last3=Steinberg | first3=C. M. | last4=Kellenberger | first4=E. | last5=Boy de la Tour | first5=E. | last6=Chevalley | first6=R. | last7=Edgar | first7=R. S. | last8=Susman | first8=M. | last9=Denhardt | first9=G. H. | last10=Lielausis | first10=A. |display-authors=5| title=Physiological Studies of Conditional Lethal Mutants of Bacteriophage T4D | journal=Cold Spring Harbor Symposia on Quantitative Biology | publisher=Cold Spring Harbor Laboratory | volume=28 | date=1963-01-01 | issn=0091-7451 | doi=10.1101/sqb.1963.028.01.053 | pages=375–394}}</ref> fundamental advances were made in our understanding of the functions and interactions of the proteins employed in the machinery of [[DNA replication]], [[DNA repair]], [[genetic recombination|DNA recombination]], and in the assembly of molecular structures.<ref>{{Cite journal |last=Edgar |first=Bob |date=2004-10-01 |title=The Genome of Bacteriophage T4 |url=https://academic.oup.com/genetics/article/168/2/575/6059485 |journal=Genetics |language=en |volume=168 |issue=2 |pages=575–582 |doi=10.1093/genetics/168.2.575 |issn=1943-2631 |pmc=1448817 |pmid=15514035}}</ref>

== Griffith's experiment ==
{{Main|Griffith's experiment}}
[[File:01 dna.jpg|thumb| Griffith's experiment]]

In 1928, [[Frederick 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. 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.<ref>{{Cite journal |last1=Ravenhall |first1=Matt |last2=Škunca |first2=Nives |last3=Lassalle |first3=Florent |last4=Dessimoz |first4=Christophe |date=May 2015 |title=Inferring Horizontal Gene Transfer |journal=PLOS Computational Biology |language=en |volume=11 |issue=5 |pages=e1004095 |doi=10.1371/journal.pcbi.1004095 |doi-access=free |pmid=26020646|pmc=4462595 |bibcode=2015PLSCB..11E4095R }}</ref>

Griffith's experiment addressed the pneumococcus 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.{{cn|date=May 2023}}

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.<ref name="Verma-2004" />

==Avery–MacLeod–McCarty experiment==
{{Main|Avery–MacLeod–McCarty experiment}}
{{AI-generated|section|date=December 2024}}
The Avery–MacLeod–McCarty experiment was a landmark study conducted in 1944 that demonstrated that DNA, not protein as previously thought, carries genetic information in bacteria. [[Oswald Avery]], [[Colin Munro MacLeod]], and [[Maclyn McCarty]] used an extract from a [[Strain (biology)|strain]] of [[pneumococcus]] that could cause [[pneumonia]] in mice. They showed that [[genetic transformation]] in the bacteria could be accomplished by injecting them with purified DNA from the extract. They discovered that when they [[Digestive enzyme|digested]] the DNA in the extract with [[DNase]], transformation of harmless bacteria into virulent ones was lost. This provided strong evidence that DNA was the genetic material, challenging the prevailing belief that proteins were responsible. It laid the basis for the subsequent discovery of its structure by Watson and Crick.

== Hershey–Chase experiment ==
{{Main|Hershey–Chase experiment}}
[[File:Hershey Chase experiment.png|thumb|Hershey–Chase experiment]]
Confirmation that DNA is the genetic material which is cause of infection came from the [[Hershey–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.<ref name="Verma-2004" />

==Meselson–Stahl experiment==
{{Main|Meselson–Stahl experiment}}
[[File:Meselson-stahl experiment diagram en chiral.svg|thumb|Meselson-Stahl experiment]]
{{AI-generated|section|date=December 2024}}
The Meselson-Stahl experiment was a landmark experiment in molecular biology that provided evidence for the [[semiconservative replication]] of DNA. Conducted in 1958 by [[Matthew Meselson]] and [[Franklin Stahl]], the experiment involved growing ''[[Escherichia coli|E. coli]]'' bacteria in a medium containing heavy isotope of nitrogen (<sup>15</sup>N) for several generations. This caused all the newly synthesized bacterial DNA to be incorporated with the heavy isotope.

After allowing the bacteria to replicate in a medium containing normal nitrogen (<sup>14</sup>N), samples were taken at various time points. These samples were then subjected to centrifugation in a density gradient, which separated the DNA molecules based on their density.

The results showed that after one generation of replication in the <sup>14</sup>N medium, the DNA formed a band of intermediate density between that of pure <sup>15</sup>N DNA and pure <sup>14</sup>N DNA. This supported the semiconservative DNA replication proposed by Watson and Crick, where each strand of the parental DNA molecule serves as a template for the synthesis of a new complementary strand, resulting in two daughter DNA molecules, each consisting of one parental and one newly synthesized strand.

The Meselson-Stahl experiment provided compelling evidence for the semiconservative replication of DNA, which is fundamental to the understanding of genetics and molecular biology.

== 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.<ref>{{Cite journal|last1=Mojiri|first1=Soheil|last2=Isbaner|first2=Sebastian|last3=Mühle|first3=Steffen|last4=Jang|first4=Hongje|last5=Bae|first5=Albert Johann|last6=Gregor|first6=Ingo|last7=Gholami|first7=Azam|last8=Gholami|first8=Azam|last9=Enderlein|first9=Jörg|date=2021-06-01|title=Rapid multi-plane phase-contrast microscopy reveals torsional dynamics in flagellar motion|url=https://www.osapublishing.org/boe/abstract.cfm?uri=boe-12-6-3169|journal=Biomedical Optics Express|language=EN|volume=12|issue=6|pages=3169–3180|doi=10.1364/BOE.419099|pmid=34221652|pmc=8221972|issn=2156-7085}}</ref> 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.<ref>{{Cite web|last=van Warmerdam|first=T.|title=Molecular Biology Laboratory Resource|url=https://www.yourbiohelper.com/research|archive-url=https://web.archive.org/web/20211229005941/https://www.yourbiohelper.com/research|url-status=usurped|archive-date=December 29, 2021|website=Yourbiohelper.com}}</ref>

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 gene editing|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.<ref>{{Cite web|last=van Warmerdam|first=T.|title=Molecular biology laboratory resource|url=https://www.yourbiohelper.com/research|archive-url=https://web.archive.org/web/20211229005941/https://www.yourbiohelper.com/research|url-status=usurped|archive-date=December 29, 2021|website=Yourbiohelper.com}}</ref>

== Relationship to other biological sciences ==
[[File:Schematic relationship between biochemistry, genetics and molecular biology.svg|thumb|Schematic relationship between [[biochemistry]], [[genetics]] and molecular biology]]

The following list describes a viewpoint on the interdisciplinary relationships between molecular biology and other related fields.<ref>{{cite book |last1=Lodish |first1=Harvey |last2=Berk |first2=Arnold |last3=Zipursky |first3=S. Lawrence |last4=Matsudaira |first4=Paul |last5=Baltimore |first5=David |last6=Darnell |first6=James | name-list-style = vanc |title=Molecular cell biology |date=2000 |publisher=Scientific American Books |location=New York |isbn=978-0-7167-3136-8 |edition=4th |url=https://archive.org/details/molecularcellbio00lodi }}</ref>
* '''''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]]s. [[Biochemist]]s focus heavily on the role, function, and structure of [[biomolecule]]s such as [[protein]]s, [[lipid]]s, [[carbohydrate]]s and [[nucleic acid]]s.<ref>{{Cite book|last=Berg|first=Jeremy|title=Biochemistry|date=2002|publisher=W.H. Freeman|others=Tymoczko, John L.; Stryer, Lubert|isbn=0-7167-3051-0|edition=5th|location=New York|oclc=48055706}}</ref>
* '''''Genetics''''' is the study of how genetic differences affect organisms. [[Genetics]] attempts to predict how [[mutation]]s, individual [[gene]]s and [[Epistasis|genetic interactions]] can affect the expression of a [[phenotype]]<ref>{{cite web|last1=Reference|first1=Genetics Home|title=Help Me Understand Genetics|url=https://ghr.nlm.nih.gov/primer|website=Genetics Home Reference|access-date=31 December 2016}}</ref>

While researchers practice techniques specific to molecular biology, it is common to combine these with methods from [[genetics]] and [[biochemistry]]. Much of molecular biology is quantitative, and recently a significant amount of work has been done using computer science techniques such as [[bioinformatics]] and [[computational biology]]. [[Molecular genetics]], 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]] and [[developmental biology]], or indirectly, where molecular techniques are used to infer historical attributes of [[population]]s or [[species]], as in fields in [[evolutionary biology]] such as [[population genetics]] and [[phylogenetics]]. There is also a long tradition of studying [[biomolecule]]s "from the ground up", or molecularly, in [[biophysics]].<ref name="Tian_2013">{{cite book| veditors = Tian J |title=Molecular Imaging: Fundamentals and Applications|url=https://books.google.com/books?id=cBXIBAAAQBAJ&pg=PA542|date=2013|publisher=Springer-Verlag Berlin & Heidelberg GmbH & Co. K|page=542|access-date=2019-07-08|isbn=9783642343032 }}</ref>

==Techniques of molecular biology==
[[File:DNA animation.gif|thumb|left|DNA animation]]
{{about||more extensive list on protein methods|protein methods|more extensive list on nucleic acid methods|nucleic acid methods}}

===Molecular cloning===
{{main|Molecular cloning}}
[[File:Transduction image.pdf|thumb|Transduction image]]
Molecular cloning is used to isolate and then transfer a DNA sequence of interest into a plasmid vector.<ref>{{Cite web|title=Foundations of Molecular Cloning - Past, Present and Future {{!}} NEB|url=https://www.neb.com/tools-and-resources/feature-articles/foundations-of-molecular-cloning-past-present-and-future|access-date=2021-11-25|website=www.neb.com}}</ref> This recombinant DNA technology was first developed in the 1960s.<ref>{{Cite web|title=Foundations of Molecular Cloning - Past, Present and Future {{!}} NEB|url=https://www.neb.com/tools-and-resources/feature-articles/foundations-of-molecular-cloning-past-present-and-future|access-date=2021-11-04|website=www.neb.com}}</ref> In this technique, a [[DNA]] sequence coding for a protein of interest is [[clone (genetics)|cloned]] using [[polymerase chain reaction]] (PCR), and/or [[restriction enzyme]]s, into a [[plasmid]] ([[expression vector]]). The plasmid vector usually has at least 3 distinctive features: an origin of replication, a [[multiple cloning site]] (MCS), and a selective marker (usually [[antibiotic resistance]]). Additionally, upstream of the MCS are the [[promoter region]]s and the [[Transcription (genetics)|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 (genetics)|transformation]] via uptake of naked DNA, [[bacterial conjugation|conjugation]] via cell-cell contact or by [[transduction (genetics)|transduction]] via viral vector. Introducing DNA into [[Eukaryote|eukaryotic]] cells, such as animal cells, by physical or chemical means is called [[transfection]]. Several different transfection techniques are available, such as calcium phosphate transfection, [[electroporation]], [[microinjection]] and [[liposome transfection]]. The plasmid may be integrated into the [[genome]], resulting in a stable transfection, or may remain independent of the genome and expressed temporarily, called a transient transfection.<ref name="cell">{{cite book|last1=Alberts|first1=Bruce|last2=Johnson|first2=Alexander|last3=Lewis|first3=Julian|last4=Raff|first4=Martin|last5=Roberts|first5=Keith|last6=Walter|first6=Peter | name-list-style = vanc |title=Isolating, Cloning, and Sequencing DNA|url=https://www.ncbi.nlm.nih.gov/books/NBK26837/|access-date=31 December 2016|language=en}}</ref><ref>{{cite book|last1=Lessard|first1=Juliane C.|title=Laboratory Methods in Enzymology: DNA|chapter=Molecular cloning|date=1 January 2013|volume=529|pages=85–98|doi=10.1016/B978-0-12-418687-3.00007-0|pmid=24011038|issn=1557-7988|isbn=978-0-12-418687-3}}</ref>

DNA coding for a protein of interest is now inside a cell, and the [[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]] can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.<ref>{{cite book|last1=Kokate |first1=Chandrakant|last2=Jalalpure|first2=Sunil S.|last3=Hurakadle|first3=Pramod J.| name-list-style = vanc |title=Textbook of Pharmaceutical Biotechnology|department=Expression Cloning|url=https://books.google.com/books?id=p70UCwAAQBAJ&pg=PA125|date=2016|publisher=Elsevier|page=125|access-date=2019-07-08|isbn=9788131239872}}</ref>

===Polymerase chain reaction===
{{main|Polymerase chain reaction}}

Polymerase chain reaction (PCR) is an extremely versatile technique for copying DNA. In brief, PCR allows a specific [[DNA sequencing|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.<ref>{{Cite journal|last=Lenstra|first=J. A.|date=July 1995|title=The applications of the polymerase chain reaction in the life sciences|url=https://pubmed.ncbi.nlm.nih.gov/7580841/|journal=Cellular and Molecular Biology (Noisy-Le-Grand, France)|volume=41|issue=5|pages=603–614|issn=0145-5680|pmid=7580841}}</ref> The PCR technique can be used to introduce [[Restriction site|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]]. 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]]) for amplification of RNA, and, more recently, [[quantitative PCR]] which allow for quantitative measurement of DNA or RNA molecules.<ref>{{cite web|title=Polymerase Chain Reaction (PCR)|url=https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/| work = National Center for Biotechnology Information | publisher = U.S. National Library of Medicine |access-date=31 December 2016}}</ref><ref>{{cite web|title=Polymerase Chain Reaction (PCR) Fact Sheet|url=https://www.genome.gov/10000207/polymerase-chain-reaction-pcr-fact-sheet/|website=National Human Genome Research Institute (NHGRI)|access-date=31 December 2016}}</ref>[[File:Two percent Agarose Gel in Borate Buffer cast in a Gel Tray (Front, angled).jpg|thumb|Two percent [[agarose|agarose gel]] in [[Borate buffered saline|borate buffer cast]] in a gel tray]]

===Gel electrophoresis===
[[File:SDS-PAGE.jpg|thumb|SDS-PAGE|left]]
{{main|Gel electrophoresis}}

Gel electrophoresis is a technique which separates molecules by their size using an agarose or polyacrylamide gel.<ref name="Lee-2012">{{Cite journal|last1=Lee|first1=Pei Yun|last2=Costumbrado|first2=John|last3=Hsu|first3=Chih-Yuan|last4=Kim|first4=Yong Hoon|date=2012-04-20|title=Agarose Gel Electrophoresis for the Separation of DNA Fragments|journal=Journal of Visualized Experiments|issue=62|pages=3923|doi=10.3791/3923|issn=1940-087X|pmc=4846332|pmid=22546956}}</ref> 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.<ref name="Lee-2012" /> Proteins can also be separated on the basis of size using an [[SDS-PAGE]] gel, or on the basis of size and their [[electric charge]] by using what is known as a [[Two-dimensional gel electrophoresis|2D gel electrophoresis]].<ref>{{cite journal | vauthors = Lee PY, Costumbrado J, Hsu CY, Kim YH | title = Agarose gel electrophoresis for the separation of DNA fragments | journal = Journal of Visualized Experiments | issue = 62 | date = April 2012 | pmid = 22546956 | pmc = 4846332 | doi = 10.3791/3923 }}</ref>
[[File:Coomassie blue stained gel.png|thumb|Proteins stained on a PAGE gel using Coomassie blue dye]]

=== The Bradford protein assay ===
{{Main|Bradford protein 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]] G-250.<ref name="Bradford-1976">{{cite journal |last1=Bradford |first1=Marion M. |title=A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding |journal=Analytical Biochemistry |date=May 1976 |volume=72 |issue=1–2 |pages=248–254 |doi=10.1016/0003-2697(76)90527-3 |pmid=942051 |s2cid=4359292 }}</ref> Coomassie Blue undergoes a visible color shift from reddish-brown to bright blue upon binding to protein.<ref name="Bradford-1976" /> In its unstable, cationic state, Coomassie Blue has a background wavelength of 465&nbsp;nm and gives off a reddish-brown color.<ref name="rufrice">{{Cite web|title=Protein determination by the Bradford method|url=https://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html|access-date=2021-11-08|website=www.ruf.rice.edu}}</ref> When Coomassie Blue binds to protein in an acidic solution, the background wavelength shifts to 595&nbsp;nm and the dye gives off a bright blue color.<ref name="rufrice" /> Proteins in the assay bind Coomassie blue in about 2 minutes, and the protein-dye complex is stable for about an hour, although it is recommended that absorbance readings are taken within 5 to 20 minutes of reaction initiation.<ref name="Bradford-1976" /> The concentration of protein in the Bradford assay can then be measured using a visible light [[Spectrophotometry|spectrophotometer]], and therefore does not require extensive equipment.<ref name="rufrice" />

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.<ref name="Bradford-1976" /> Unlike the previous methods, the Bradford assay is not susceptible to interference by several non-protein molecules, including ethanol, sodium chloride, and magnesium chloride.<ref name="Bradford-1976" /> However, it is susceptible to influence by strong alkaline buffering agents, such as [[sodium dodecyl sulfate]] (SDS).<ref name="Bradford-1976" />

===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]]ting'', after the technique described by [[Edwin Southern]] for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as the ''northern blot'', actually did not use the term.<ref>{{cite journal | vauthors = Thomas PS | title = Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 77 | issue = 9 | pages = 5201–5 | date = September 1980 | pmid = 6159641 | pmc = 350025 | doi = 10.1073/pnas.77.9.5201 | bibcode = 1980PNAS...77.5201T | doi-access = free }}</ref>

====Southern blotting====
{{main|Southern blot}}

Named after its inventor, biologist [[Edwin Southern]], 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]] (restriction endonuclease) digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via [[capillary action]]. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest.<ref>{{cite journal |doi=10.1002/0471142735.im1006as06 |title=Southern Blotting |date=1993 |last1=Brown |first1=Terry |journal=Current Protocols in Immunology |volume=6 |pages=Unit 10.6A |pmid=18432697 }}</ref> Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as [[Polymerase chain reaction|PCR]], to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring [[transgene]] copy number in [[Genetically modified organism|transgenic mice]] or in the engineering of [[gene knockout]] [[Stem cell line|embryonic stem cell lines]].<ref name="Tian_2013"/>

====Northern blotting====
{{main|Northern blot}}
[[File:Northern blot diagram.png|thumb|Northern blot diagram]]
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.<ref>{{cite book |doi=10.1007/978-1-59745-248-9_7 |chapter=Northern Blotting Analysis |title=RNA |series=Methods in Molecular Biology |date=2011 |last1=Josefsen |first1=Knud |last2=Nielsen |first2=Henrik |volume=703 |pages=87–105 |pmid=21125485 |isbn=978-1-58829-913-0 }}</ref><ref>{{cite book | vauthors = He SL, Green R | title = Laboratory Methods in Enzymology: RNA | chapter = Northern blotting | volume = 530 | pages = 75–87 | date = 1 January 2013 | pmid = 24034315 | pmc = 4287216 | doi = 10.1016/B978-0-12-420037-1.00003-8 | isbn = 978-0-12-420037-1 }}</ref>

====Western blotting====
{{main|Western blot}}
A western blot is a technique by which specific proteins can be detected from a mixture of proteins.<ref name="Mahmood-2012" /> Western blots can be used to determine the size of isolated proteins, as well as to quantify their expression.<ref>{{Cite web|title=Western blot {{!}} Learn Science at Scitable|url=https://www.nature.com/scitable/definition/western-blot-288/|access-date=2021-11-25|website=www.nature.com|language=en}}</ref> In [[western blot]]ting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as [[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.<ref name="Mahmood-2012">{{cite journal | vauthors = Mahmood T, Yang PC | title = Western blot: technique, theory, and trouble shooting | journal = North American Journal of Medical Sciences | volume = 4 | issue = 9 | pages = 429–34 | date = September 2012 | doi = 10.4103/1947-2714.100998 | doi-broken-date = 1 November 2024 | doi-access = free | pmid = 23050259 | pmc = 3456489 }}</ref><ref>{{cite journal | vauthors = Kurien BT, Scofield RH | title = Western blotting | journal = Methods | volume = 38 | issue = 4 | pages = 283–93 | date = April 2006 | pmid = 16483794 | doi = 10.1016/j.ymeth.2005.11.007 }}</ref>

====Eastern blotting====
{{main|Eastern blot}}

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.<ref>{{cite journal | vauthors = Thomas S, Thirumalapura N, Crossley EC, Ismail N, Walker DH | title = Antigenic protein modifications in Ehrlichia | journal = Parasite Immunology | volume = 31 | issue = 6 | pages = 296–303 | date = June 2009 | pmid = 19493209 | pmc = 2731653 | doi = 10.1111/j.1365-3024.2009.01099.x }}</ref>

===Microarrays===
{{main|DNA microarray}}
[[Image:Microarray printing.ogv|thumb|left|A DNA microarray being printed]]
[[File:NA hybrid.svg|thumb|Hybridization of target to probe]]
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 sequencing|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.<ref>{{cite web|title=Microarrays|url=https://www.ncbi.nlm.nih.gov/probe/docs/techmicroarray/| work  = National Center for Biotechnology Information | publisher = U.S. National Library of Medicine |access-date=31 December 2016}}</ref><ref>{{cite journal |doi=10.1002/0471142727.mb2201s101 |title=Overview of DNA Microarrays: Types, Applications, and Their Future |date=2013 |last1=Bumgarner |first1=Roger |journal=Current Protocols in Molecular Biology |volume=101 |pages=Unit 22.1 |pmid=23288464 |pmc=4011503 }}</ref><ref>{{cite journal | vauthors = Govindarajan R, Duraiyan J, Kaliyappan K, Palanisamy M | title = Microarray and its applications | journal = Journal of Pharmacy & Bioallied Sciences | volume = 4 | issue = Suppl 2 | pages = S310-2 | date = August 2012 | pmid = 23066278 | pmc = 3467903 | doi = 10.4103/0975-7406.100283 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Tarca AL, Romero R, Draghici S | title = Analysis of microarray experiments of gene expression profiling | journal = American Journal of Obstetrics and Gynecology | volume = 195 | issue = 2 | pages = 373–88 | date = August 2006 | pmid = 16890548 | pmc = 2435252 | doi = 10.1016/j.ajog.2006.07.001 }}</ref>

===Allele-specific oligonucleotide===
{{Main|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 unhybridized probes 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.<ref>{{cite book|editor-first2= David Y.|editor-last2=Zhang|editor-last1= Cheng|editor-first1=Liang | name-list-style = vanc |title=Molecular genetic pathology|date=2008|publisher=Humana|location=Totowa, NJ|isbn=978-1-59745-405-6|page=96|url=https://books.google.com/books?id=F_7QXO0ZBigC&q=Allele-specific+oligonucleotide&pg=PA97|access-date=31 December 2016|language=en}}</ref><ref>{{cite book|last1=Leonard|first1=Debra G.B. | name-list-style = vanc |title=Molecular Pathology in Clinical Practice|date=2016|publisher=Springer|isbn=978-3-319-19674-9|page=31|url=https://books.google.com/books?id=cDWFCwAAQBAJ&q=Allele-specific+oligonucleotide&pg=PA30|access-date=31 December 2016|language=en}}</ref>

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.<ref>{{cite book | veditors = Tian J |title=Molecular Imaging: Fundamentals and Applications|url=https://books.google.com/books?id=cBXIBAAAQBAJ&pg=PA550|date=2013|publisher=Springer-Verlag Berlin & Heidelberg GmbH & Co.K |pages=550, 552|access-date=2019-07-08|isbn=9783642343032 }}</ref>

== See also ==
{{Columns-list|colwidth=30em|
* [[Astrobiology]]
* [[Central dogma of molecular biology]]
* [[Genetic code]]
* [[Geniom RT Analyzer]], diagnostic testing instrument
* [[Genome]]
* [[:Category:Molecular biology institutes|Molecular biology institutes]]
* [[Molecular engineering]]
* [[Molecular modeling]]
* [[Protein–protein interaction prediction|Protein interaction prediction]]
* [[Protein structure prediction]]
* [[Proteome]]
* [[Cell biology]]
}}

== References ==
{{reflist}}

== Further reading ==
{{refbegin}}
* {{cite journal | vauthors = Cohen SN, Chang AC, Boyer HW, Helling RB | title = Construction of biologically functional bacterial plasmids in vitro | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 70 | issue = 11 | pages = 3240–4 | date = November 1973 | pmid = 4594039 | pmc = 427208 | doi = 10.1073/pnas.70.11.3240 | bibcode = 1973PNAS...70.3240C | doi-access = free }}
* {{cite magazine | vauthors = Rodgers M | title = The Pandora's box congress. | magazine = Rolling Stone | volume = 189 | pages = 37–77 | date = June 1975 }}
* {{cite book | first1= Keith | last1 = Roberts | first2 = Martin | last2 = Raff | first3 = Bruce | last3 = Alberts | first4 = Peter | last4 = Walter | first5 = Julian | last5 = Lewis | first6 = Alexander | last6 = Johnson | name-list-style = vanc | title = Molecular Biology of the Cell | url = https://www.ncbi.nlm.nih.gov/books/NBK21054/ | isbn = 978-0-8153-3218-3 | publisher = Garland Science | year = 2002 }}
{{refend}}

== External links ==
{{Library resources box}}
* {{Commons category-inline}}
<!--========================({{No More Links}})============================
    | PLEASE BE CAUTIOUS IN ADDING MORE LINKS TO THIS ARTICLE. WIKIPEDIA  |
    | IS NOT A COLLECTION OF LINKS NOR SHOULD IT BE USED FOR ADVERTISING. |
    |                                                                     |
    |           Excessive or inappropriate links WILL BE DELETED.         |
    | See [[Wikipedia:External links]] & [[Wikipedia:Spam]] for details.  |
    |                                                                     |
    | If there are already plentiful links, please propose additions or   |
    | replacements on this article's discussion page, or submit your link |
    | to the relevant category at the "long dead (2017)" Open Directory Project (dmoz.org)   |
    | and link back to that category using the {{dmoz}} template.         |
    =======================({{No More Links}})=============================-->

{{Molecular biology}}
{{Molecular and cellular biology}}{{Biochemistry topics}}
{{Gene expression}}
{{Branches of biology}}
{{Biotechnology}}
{{Branches of chemistry}}
{{Authority control}}
{{Portal bar|Biology}}

[[Category:Molecular biology| ]]
[[Category:Applied geometry]]