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104-458: In molecular biology lin-4 is a microRNA (miRNA) that was identified from a study of developmental timing in the nematode Caenorhabditis elegans . It was the first to be discovered of the miRNAs, a class of non-coding RNAs involved in gene regulation. miRNAs are transcribed as ~70 nucleotide precursors and subsequently processed by the Dicer enzyme to give a 21 nucleotide product. The extents of

208-446: A 2D gel electrophoresis . 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 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

312-461: A form of gene augmentation where new gene is added, may improve a cells function without modifying the genes that cause a disorder. Gene therapy may be classified into two types by the type of cell it affects: somatic cell and germline gene therapy. In somatic cell gene therapy (SCGT), the therapeutic genes are transferred into any cell other than a gamete , germ cell , gametocyte , or undifferentiated stem cell . Any such modifications affect

416-736: 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 regions and the transcription start site, which regulate the expression of cloned gene. This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation via uptake of naked DNA, conjugation via cell-cell contact or by transduction via viral vector. Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means

520-450: A retrovirus , can modify a cell's nuclear genome to permanently express a gene, although vectors can be modified to prevent integration. Retroviruses were used in 18% of trials before 2018. Libmeldy is an ex vivo stem cell treatment for metachromatic leukodystrophy which uses a lentiviral vector and was approved by the European medical agency in 2020. Adeno-associated virus (AAV)

624-447: A cancer cell to die. Another approach is the use oncolytic viruses , such as Oncorine, which are viruses that selectively reproduce in cancerous cells leaving other cells unaffected. mRNA has been suggested as a non-viral vector for cancer gene therapy that would temporarily change a cancerous cell's function to create antigens or kill the cancerous cells and there have been several trials. Afamitresgene autoleucel , sold under

728-546: A cell without permanently altering the cell's nuclear genome. These vectors can be used to cause antigens to be added to cancers causing an immune response, or hinder angiogenesis by expressing certain proteins. An Adenovirus vector is used in the commercial products Gendicine and Oncorine . Another commercial product, Rexin G , uses a retrovirus-based vector and selectively binds to receptors that are more expressed in tumors. One approach, suicide gene therapy , works by introducing genes encoding enzymes that will cause

832-462: A cell's genetic expression with genetic material that is not integrated into the host cell's DNA. As of 2017, such vectors were used in 20% of trials for gene therapy. Adenovirus vectors are mostly used in cancer treatments and novel genetic vaccines such as the Ebola vaccine , vaccines used in clinical trials for HIV and SARS-CoV-2 , or cancer vaccines . Lentiviral vectors based on lentivirus ,

936-480: A cell. Fact-checkers , such as Full Fact , Reuters , PolitiFact , and FactCheck.org said that calling the vaccines a gene therapy was incorrect. Podcast host Joe Rogan was criticized for calling mRNA vaccines gene therapy as was British politician Andrew Bridgen , with fact checker Full Fact calling for Bridgen to be removed from the conservative party for this and other statements. Gene therapy encapsulates many forms of adding different nucleic acids to

1040-402: A cell. Gene augmentation adds a new protein coding gene to a cell. One form of gene augmentiation is gene replacement therapy , a treatment for monogenic recessive disorders where a single gene is not functional an additional functional gene is added. For diseases caused by multiple genes or a dominant gene, gene silencing or gene editing approaches are more appropriate but gene addition,

1144-412: A chimeric antigen receptor. In vivo gene therapy is seen as simpler, since it does not require the harvesting of mitotic cells. However, ex vivo gene therapies are better tolerated and less associated with severe immune responses. The death of Jesse Gelsinger in a trial of an adenovirus -vectored treatment for ornithine transcarbamylase deficiency due to a systemic inflammatory reaction led to

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1248-465: A chromosome and returning the transformed cells to patients. Gene editing is a potential approach to alter the human genome to treat genetic diseases, viral diseases, and cancer. As of 2020 these approaches are being studied in clinical trials. In 1986, a meeting at the Institute Of Medicine defined gene therapy as the addition or replacement of a gene in a targeted cell type. In

1352-405: A copy of this gene that does not contain the deleterious mutation and thereby produces a functional protein. This strategy is referred to as gene replacement therapy and could be employed to treat inherited retinal diseases. While the concept of gene replacement therapy is mostly suitable for recessive diseases, novel strategies have been suggested that are capable of also treating conditions with

1456-493: A defect in lipoprotein lipase . DNA must be administered, reach the damaged cells, enter the cell and either express or disrupt a protein. Multiple delivery techniques have been explored. The initial approach incorporated DNA into an engineered virus to deliver the DNA into a chromosome . Naked DNA approaches have also been explored, especially in the context of vaccine development. Generally, efforts focused on administering

1560-402: A density gradient, which separated the DNA molecules based on their density. The results showed that after one generation of replication in the N medium, the DNA formed a band of intermediate density between that of pure N DNA and pure 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

1664-727: A dominant pattern of inheritance. In vivo, gene editing systems using CRISPR have been used in studies with mice to treat cancer and have been effective at reducing tumors. In vitro, the CRISPR system has been used to treat HPV cancer tumors. Adeno-associated virus , Lentivirus based vectors have been to introduce the genome for the CRISPR system. The delivery of DNA into cells can be accomplished by multiple methods . The two major classes are recombinant viruses (sometimes called biological nanoparticles or viral vectors) and naked DNA or DNA complexes (non-viral methods). In order to replicate , viruses introduce their genetic material into

1768-723: A functional, therapeutic gene to replace a mutated gene. The polymer molecule is packaged within a " vector ", which carries the molecule inside cells. Early clinical failures led to dismissals of gene therapy. Clinical successes since 2006 regained researchers' attention, although as of 2014 , it was still largely an experimental technique. These include treatment of retinal diseases Leber's congenital amaurosis and choroideremia , X-linked SCID , ADA-SCID, adrenoleukodystrophy , chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), multiple myeloma , haemophilia , and Parkinson's disease . Between 2013 and April 2014, US companies invested over $ 600 million in

1872-412: A gene that causes a needed protein to be expressed. More recently, increased understanding of nuclease function has led to more direct DNA editing, using techniques such as zinc finger nucleases and CRISPR . The vector incorporates genes into chromosomes. The expressed nucleases then knock out and replace genes in the chromosome. As of 2014 these approaches involve removing cells from patients, editing

1976-487: A genetic disorder or the replacement of multiple genes is not yet possible. Only a few of the trials are in the advanced stages. In germline gene therapy (GGT), germ cells ( sperm or egg cells ) are modified by the introduction of functional genes into their genomes. Modifying a germ cell causes all the organism's cells to contain the modified gene. The change is therefore heritable and passed on to later generations. Australia, Canada, Germany, Israel, Switzerland, and

2080-402: A healthy gene have been proposed and are being studied for treating some genetic diseases. As of 2017, 11.1% of gene therapy clinical trials targeted monogenic diseases. Diseases such as sickle cell disease that are caused by autosomal recessive disorders for which a person's normal phenotype or cell function may be restored in cells that have the disease by a normal copy of the gene that

2184-526: 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. The Avery–MacLeod–McCarty experiment

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2288-453: A labeled 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

2392-446: A micropipette directly into a living mammalian cell, and exposing cells to a precipitate of DNA that contained the desired genes. Scientists theorized that a virus could also be used as a vehicle, or vector, to deliver new genes into cells. One of the first scientists to report the successful direct incorporation of functional DNA into a mammalian cell was biochemist Dr. Lorraine Marquardt Kraus (6 September 1922 – 1 July 2016) at

2496-518: A mixture of proteins. Western blots can be used to determine the size of isolated proteins, as well as to quantify their expression. In western blotting , proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE . 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 antibodies . Antibodies that specifically bind to

2600-439: A number of laboratories are working on delivery of PEGylated particles, which form serum protein coronas and chiefly exhibit LDL receptor mediated uptake in cells in vivo . There have been attempts to treat cancer using gene therapy. As of 2017, 65% of gene therapy trials were for cancer treatment. Adenovirus vectors are useful for some cancer gene therapies because adenovirus can transiently insert genetic material into

2704-452: A patient themselves, but include ex vivo approaches, and would not depend on the vector used. During the COVID-19 pandemic, some academics insisted that the mRNA vaccines for COVID were not gene therapy to prevent the spread of incorrect information that the vaccine could alter DNA, other academics maintained that the vaccines were a gene therapy because they introduced genetic material into

2808-405: A patient's genetic makeup can be considered gene therapy. Bone marrow transplantation and organ transplants in general have been found to introduce foreign DNA into patients. Gene therapy was first conceptualized in the 1960s, when the feasibility of adding new genetic functions to mammalian cells began to be researched. Several methods to do so were tested, including injecting genes with

2912-413: A potential application of AAV gene therapy for human disease. Non-viral vectors for gene therapy present certain advantages over viral methods, such as large scale production and low host immunogenicity . However, non-viral methods initially produced lower levels of transfection and gene expression , and thus lower therapeutic efficacy. Newer technologies offer promise of solving these problems, with

3016-436: 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 is recommended that absorbance readings are taken within 5 to 20 minutes of reaction initiation. The concentration of protein in

3120-508: A resultant retardation of developmental fates, from their initially early to instead later larval stages. Consequences include heterochronic developmental patterns, with loss of structures such as the vulva and adult cuticle. lin-4 acts as a negative regulator of lin-14 and represses accumulation of the LIN-14 protein. It also targets lin-28 and reduces its protein expression through translational inhibition. The base pairing in place between lin-4 and

3224-408: A single slide. Each spot has a DNA fragment molecule that is complementary to a single 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

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3328-584: A success was demonstrated in a trial that started on 14 September 1990, when Ashanthi DeSilva was treated for ADA - SCID . The first somatic treatment that produced a permanent genetic change was initiated in 1993. The goal was to cure malignant brain tumors by using recombinant DNA to transfer a gene making the tumor cells sensitive to a drug that in turn would cause the tumor cells to die. The polymers are either translated into proteins , interfere with target gene expression , or possibly correct genetic mutations . The most common form uses DNA that encodes

3432-416: A temporary blueprint that degrades naturally, as in a non-integrative vectors , or to enter the host's nucleus becoming a permanent part of the host's nuclear DNA in infected cells. A number of viruses have been used for human gene therapy, including viruses such as lentivirus , adenoviruses , herpes simplex , vaccinia , and adeno-associated virus . Adenovirus viral vectors (Ad) temporarily modify

3536-460: A temporary halt on gene therapy trials across the United States. As of 2021 , in vivo and ex vivo therapeutics are both seen as safe. The concept of gene therapy is to fix a genetic problem at its source. If, for instance, a mutation in a certain gene causes the production of a dysfunctional protein resulting (usually recessively) in an inherited disease, gene therapy could be used to deliver

3640-462: A viewpoint on the interdisciplinary relationships between molecular biology and other related fields. 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 ,

3744-612: Is a medical technology that aims to produce a therapeutic effect through the manipulation of gene expression or through altering the biological properties of living cells. The first attempt at modifying human DNA was performed in 1980, by Martin Cline , but the first successful nuclear gene transfer in humans, approved by the National Institutes of Health , was performed in May 1989. The first therapeutic use of gene transfer as well as

3848-408: Is a virus that is incapable of transmission between cells unless the cell is infected by another virus, a helper virus. Adenovirus and the herpes viruses act as helper viruses for AAV. AAV persists within the cell outside of the cell's nuclear genome for an extended period of time through the formation of concatemers mostly organized as episomes . Genetic material from AAV vectors is integrated into

3952-439: Is also a long tradition of studying biomolecules "from the ground up", or molecularly, in biophysics . 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 using polymerase chain reaction (PCR), and/or restriction enzymes , into

4056-439: 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. The following list describes

4160-413: 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. DNA coding for a protein of interest

4264-410: 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

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4368-433: 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. 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

4472-405: Is introduced to the patient, which then achieves the desired biological effect by passing the genetic material (e.g. for a missing protein) into the patient's cells. In ex vivo gene therapies, such as CAR-T therapeutics, the patient's own cells (autologous) or healthy donor cells (allogeneic) are modified outside the body (hence, ex vivo ) using a vector to express a particular protein, such as

4576-454: Is known as horizontal gene transfer (HGT). This phenomenon is now referred to as genetic transformation. 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,

4680-431: Is mutated, may be a good candidate for gene therapy treatment. The risks and benefits related to gene therapy for sickle cell disease are not known. Gene therapy has been used in the eye . The eye is especially suitable for adeno-associated virus vectors. Voretigene neparvovec is an approved gene therapy to treat Leber's hereditary optic neuropathy . alipogene tiparvovec , a treatment for pancreatitis caused by

4784-472: 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

4888-456: 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. A western blot is a technique by which specific proteins can be detected from

4992-458: Is seen to take up an unusual kinked structure, caused by induced changes in the groove dimension and base stacking due to the mismatched base pairs. This lin-4:lin-14 pair have been linked to the IGF-1 pathway in C. elegans with regards to their modification of development. lin-4 acts to dictate the onset of larval stages of developmental events in C. elegans . lin-4 loss of function (lf) mutations see

5096-479: Is susceptible to influence by strong alkaline buffering agents, such as sodium dodecyl sulfate (SDS). 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 for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as

5200-400: 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 gel, or on the basis of size and their electric charge by using what is known as

5304-412: 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 copy number in transgenic mice or in

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5408-516: 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. 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

5512-553: The 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

5616-515: The National Institutes of Health (NIH), Bethesda, in the United States successfully corrected genetic defects associated with Lesch-Nyhan syndrome , a debilitating neurological disease , by adding foreign DNA to cultured cells collected from patients suffering from the disease. The first attempt, an unsuccessful one, at gene therapy (as well as the first case of medical transfer of foreign genes into humans not counting organ transplantation )

5720-526: The University of Tennessee Health Science Center in Memphis, Tennessee . In 1961, she managed to genetically alter the hemoglobin of cells from bone marrow taken from a patient with sickle cell anaemia . She did this by incubating the patient's cells in tissue culture with DNA extracted from a donor with normal hemoglobin . In 1968, researchers Theodore Friedmann , Jay Seegmiller, and John Subak-Sharpe at

5824-425: 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, fundamental advances were made in our understanding of

5928-422: The northern blot , actually did not use the term. 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

6032-495: The viral capsid , lower immunogenicity, ability to transduce both dividing and nondividing cells, the potential to integrate site specifically and to achieve long-term expression in the in-vivo treatment. ASO / siRNA approaches such as those conducted by Alnylam and Ionis Pharmaceuticals require non-viral delivery systems, and utilize alternative mechanisms for trafficking to liver cells by way of GalNAc transporters. Not all medical procedures that introduce alterations to

6136-631: 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

6240-456: The DNA model was Phoebus Levene , who proposed the "polynucleotide model" of DNA in 1919 as a result of his biochemical experiments on yeast. 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. Second, the total concentration of purines (adenine and guanine) is always equal to

6344-604: The Journal of Law and the Biosciences, Sherkow et al. argued for a narrower definition of gene therapy than the FDA's in light of new technology that would consist of any treatment that intentionally and permanently modified a cell's genome, with the definition of genome including episomes outside the nucleus but excluding changes due to episomes that are lost over time. This definition would also exclude introducing cells that did not derive from

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6448-489: The L2 stage of post-embryonic development. Additional to this is the up-regulation of endogenous lin-4 primary transcripts upon appearance of the lin-4 mature form. lin-4 is found on chromosome II in C. elegans and is complementary to sequences in the 3' untranslated region (UTR) of lin-14 mRNA, this complementary transcript containing seven binding sites along with the 9 nucleotide core element CUCAGGGAA. The lin-4: lin-14 duplex

6552-457: The Netherlands prohibit GGT for application in human beings, for technical and ethical reasons, including insufficient knowledge about possible risks to future generations and higher risks versus SCGT. The US has no federal controls specifically addressing human genetic modification (beyond FDA regulations for therapies in general). In in vivo gene therapy, a vector (typically, a virus)

6656-485: The advent of increased cell-specific targeting and subcellular trafficking control. Methods for non-viral gene therapy include the injection of naked DNA, electroporation , the gene gun , sonoporation , magnetofection , the use of oligonucleotides , lipoplexes, dendrimers, and inorganic nanoparticles. These therapeutics can be administered directly or through scaffold enrichment . More recent approaches, such as those performed by companies such as Ligandal , offer

6760-437: 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;

6864-473: 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

6968-403: 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

7072-673: 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. In 1953, Francis Crick , James Watson , Rosalind Franklin , and their colleagues at

7176-410: The brand name Tecelra, is an autologous T cell immunotherapy used for the treatment of synovial sarcoma . It is a T cell receptor (TCR) gene therapy. It is the first FDA-approved engineered cell therapy for a solid tumor. It uses a self-inactivating lentiviral vector to express a T-cell receptor specific for MAGE-A4, a melanoma-associated antigen. Gene therapy approaches to replace a faulty gene with

7280-493: 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 from attempts to understand the set of rules underlying reproduction and heredity , and the nature of the hypothetical units of heredity known as genes . Gregor Mendel pioneered this work in 1866, when he first described

7384-401: The engineering of gene knockout embryonic stem cell lines . 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 RNA gel electrophoresis , and a blot . In this process RNA is separated based on size and is then transferred to a membrane that is then probed with

7488-412: The experiment involved growing E. coli bacteria in a medium containing heavy isotope of nitrogen ( 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 ( N), samples were taken at various time points. These samples were then subjected to centrifugation in

7592-399: The extract. They discovered that when they 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. Confirmation that DNA is

7696-518: The field. The first commercial gene therapy, Gendicine , was approved in China in 2003, for the treatment of certain cancers. In 2011, Neovasculgen was registered in Russia as the first-in-class gene-therapy drug for treatment of peripheral artery disease , including critical limb ischemia . In 2012, alipogene tiparvovec , a treatment for a rare inherited disorder , lipoprotein lipase deficiency , became

7800-956: The first direct insertion of human DNA into the nuclear genome was performed by French Anderson in a trial starting in September 1990. Between 1989 and December 2018, over 2,900 clinical trials were conducted, with more than half of them in phase I . In 2003, Gendicine became the first gene therapy to receive regulatory approval. Since that time, further gene therapy drugs were approved, such as alipogene tiparvovec (2012), Strimvelis (2016), tisagenlecleucel (2017), voretigene neparvovec (2017), patisiran (2018), onasemnogene abeparvovec (2019), idecabtagene vicleucel (2021), nadofaragene firadenovec , valoctocogene roxaparvovec and etranacogene dezaparvovec (all 2022). Most of these approaches utilize adeno-associated viruses (AAVs) and lentiviruses for performing gene insertions, in vivo and ex vivo , respectively. AAVs are characterized by stabilizing

7904-727: The first treatment to be approved for clinical use in either the European Union or the United States after its endorsement by the European Commission . Following early advances in genetic engineering of bacteria, cells, and small animals, scientists started considering how to apply it to medicine. Two main approaches were considered – replacing or disrupting defective genes. Scientists focused on diseases caused by single-gene defects, such as cystic fibrosis , haemophilia, muscular dystrophy , thalassemia , and sickle cell anemia . alipogene tiparvovec treats one such disease, caused by

8008-504: The functions and interactions of the proteins employed in the machinery of DNA replication , DNA repair , DNA recombination , and in the assembly of molecular structures. 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

8112-526: 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

8216-432: The hairpin precursors are not generally known and are estimated based on hairpin prediction. The products are thought to have regulatory roles through complete or partial complementarity to mRNA. The lin-4 gene has been found to lie within a 4.11kb intron of a separate host gene (see also [1] ). lin-4 is transcribed from autonomous miRNA promoters and is developmentally regulated, with lin-4 miRNA accumulation occurring at

8320-499: The host cell's nuclear genome at a low frequency and likely mediated by the DNA-modifying enzymes of the host cell. Animal models suggest that integration of AAV genetic material into the host cell's nuclear genome may cause hepatocellular carcinoma , a form of liver cancer . Several AAV investigational agents have been explored in treatment of wet age related macular degeneration by both intravitreal and subretinal approaches as

8424-441: The host cell, tricking the host's cellular machinery into using it as blueprints for viral proteins. Retroviruses go a stage further by having their genetic material copied into the nuclear genome of the host cell. Scientists exploit this by substituting part of a virus's genetic material with therapeutic DNA or RNA. Like the genetic material (DNA or RNA) in viruses, therapeutic genetic material can be designed to simply serve as

8528-510: The implications of this unique structure for possible mechanisms of DNA replication. 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. 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. Thus

8632-605: The individual patient only, and are not inherited by offspring . Somatic gene therapy represents mainstream basic and clinical research, in which therapeutic DNA (either integrated in the genome or as an external episome or plasmid ) is used to treat disease. Over 600 clinical trials utilizing SCGT are underway in the US. Most focus on severe genetic disorders, including immunodeficiencies , haemophilia , thalassaemia , and cystic fibrosis . Such single gene disorders are good candidates for somatic cell therapy. The complete correction of

8736-434: The laws of inheritance he observed in his studies of mating crosses in pea plants. One such law of genetic inheritance is the law of segregation , which states that diploid individuals with two alleles 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 . A major milestone in molecular biology

8840-415: 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 (ASO) is a technique that allows detection of single base mutations without

8944-399: 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

9048-1037: The non-viral gene delivery problem. Non-viral techniques offer the possibility of repeat dosing and greater tailorability of genetic payloads, which in the future will be more likely to take over viral-based delivery systems. Companies such as Editas Medicine , Intellia Therapeutics , CRISPR Therapeutics , Casebia , Cellectis , Precision Biosciences , bluebird bio , Excision BioTherapeutics , and Sangamo have developed non-viral gene editing techniques, however frequently still use viruses for delivering gene insertion material following genomic cleavage by guided nucleases . These companies focus on gene editing, and still face major delivery hurdles. BioNTech , Moderna Therapeutics and CureVac focus on delivery of mRNA payloads, which are necessarily non-viral delivery problems. Alnylam , Dicerna Pharmaceuticals , and Ionis Pharmaceuticals focus on delivery of siRNA (antisense oligonucleotides) for gene suppression, which also necessitate non-viral delivery systems. In academic contexts,

9152-463: The pharmaceutical industry, the activity of new drugs against the protein can be studied. Polymerase chain reaction (PCR) is an extremely versatile technique for copying DNA. In brief, PCR allows a specific 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

9256-434: The possibility of creating cell-specific targeting technologies for a variety of gene therapy modalities, including RNA, DNA and gene editing tools such as CRISPR. Other companies, such as Arbutus Biopharma and Arcturus Therapeutics , offer non-viral, non-cell-targeted approaches that mainly exhibit liver trophism. In more recent years, startups such as Sixfold Bio , GenEdit , and Spotlight Therapeutics have begun to solve

9360-405: 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 or polyacrylamide ), the size of DNA molecules

9464-530: 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 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 cells or tissue sections. The eastern blotting technique

9568-515: The same year, the FDA announced that it had jurisdiction over approving "gene therapy" without defining the term. The FDA added a very broad definition in 1993 of any treatment that would 'modify or manipulate the expression of genetic material or to alter the biological properties of living cells'. In 2018 this was narrowed to 'products that mediate their effects by transcription or translation of transferred genetic material or by specifically altering host (human) genetic sequences'. Writing in 2018, in

9672-421: 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 . PCR can also be used to determine whether a particular DNA fragment

9776-532: 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 populations or species , as in fields in evolutionary biology such as population genetics and phylogenetics . There

9880-554: 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. 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

9984-451: The target In question is discontinuous, consisting of two short helices. Molecular biology 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

10088-482: 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. 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 ,

10192-447: 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, based on the X-ray crystallography work done by Rosalind Franklin which was conveyed to them by Maurice Wilkins and Max Perutz . Watson and Crick described the structure of DNA and conjectured about

10296-443: The use of molecular biology or molecular cell biology in medicine is now referred to as molecular medicine . 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

10400-467: 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 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

10504-495: Was conveyed to them by Maurice Wilkins and Max Perutz . Their work led to the discovery of DNA in other microorganisms, plants, and animals. The field of molecular biology includes techniques which enable scientists to learn about molecular processes. These techniques are used to efficiently target new drugs, diagnose disease, and better understand cell physiology. Some clinical research and medical therapies arising from molecular biology are covered under gene therapy , whereas

10608-485: Was performed by geneticist Martin Cline of the University of California, Los Angeles in California , United States on 10 July 1980. Cline claimed that one of the genes in his patients was active six months later, though he never published this data or had it verified. After extensive research on animals throughout the 1980s and a 1989 bacterial gene tagging trial on humans, the first gene therapy widely accepted as

10712-414: 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. 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

10816-433: Was typically determined by rate sedimentation in 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. Gene therapy Gene therapy

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