Editing Molecular biology (section)

==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>