The lysogenic lifecycle begins once the cI protein reaches a high enough concentration to activate its promoters, after a small number of infections.


The prophage is duplicated with every subsequent cell division of the host. The phage genes expressed in this dormant state code for proteins that repress expression of other phage genes (such as the structural and lysis genes) in order to prevent entry into the lytic cycle. These repressive proteins are broken down when the host cell is under stress, resulting in the expression of the repressed phage genes. Stress can be from  starvation ,  poisons  (like  antibiotics ), or other factors that can damage or destroy the host. In response to stress, the activated prophage is excised from the DNA of the host cell by one of the newly expressed gene products and enters its lytic pathway.


The integration of phage λ takes place at a special attachment site in the bacterial and phage genomes, called  att λ . The sequence of the bacterial att site is called  attB , between the  gal  and  bio  operons, and consists of the parts B-O-B', whereas the complementary sequence in the circular phage genome is called  attP  and consists of the parts P-O-P'. The integration itself is a sequential exchange (see  genetic recombination ) via a  Holliday junction  and requires both the phage protein Int and the bacterial protein IHF ( integration host factor ). Both Int and IHF bind to  attP  and form an intasome, a DNA-protein-complex designed for  site-specific recombination  of the phage and host DNA. The original B-O-B' sequence is changed by the integration to B-O-P'-phage DNA-P-O-B'. The phage DNA is now part of the host's genome. [ 19 ]


The classic induction of a lysogen involved irradiating the infected cells with UV light. Any situation where a lysogen undergoes DNA damage or the  SOS response  of the host is otherwise stimulated leads to induction.


Multiplicity reactivation (MR) is the process by which multiple viral genomes, each containing inactivating genome damage, interact within an infected cell to form a viable viral genome. MR was originally discovered with phage T4, but was subsequently found in phage λ (as well as in numerous other bacterial and mammalian viruses [ 20 ] ).  MR of phage λ inactivated by UV light depends on the recombination function of either the host or of the infecting phage. [ 21 ]   Absence of both recombination systems leads to a loss of MR.


Survival of UV-irradiated phage λ is increased when the E. coli host is lysogenic for an homologous prophage, a phenomenon termed prophage reactivation. [ 22 ]   Prophage reactivation in phage λ appears to occur by a recombinational repair process similar to that of MR.


The  repressor  found in the phage lambda is a notable example of the level of control possible over gene expression by a very simple system. It forms a 'binary switch' with two genes under mutually exclusive expression, as discovered by  Barbara J. Meyer . [ 23 ]


The lambda repressor gene system consists of (from left to right on the chromosome):


The lambda repressor is a self assembling dimer also known as the cI protein. [ 24 ]  It binds DNA in the helix-turn-helix binding motif. It regulates the transcription of the cI protein and the Cro protein.


The life cycle of lambda phages is controlled by cI and Cro proteins. The lambda phage will remain in the lysogenic state if cI proteins predominate, but will be transformed into the lytic cycle if cro proteins predominate.


The cI dimer may bind to any of three operators, O R 1, O R 2, and O R 3, in the order O R 1 > O R 2 > O R 3.
Binding of a cI dimer to O R 1 enhances binding of a second cI dimer to O R 2, an effect called  cooperativity . Thus,  O R 1 and O R 2 are almost always simultaneously occupied by cI. However, this does not increase the affinity between cI and O R 3, which will be occupied only when the cI concentration is high.


At high concentrations of cI, the dimers will also bind to operators O L 1 and O L 2 (which are over 2 kb downstream from the R operators). When cI dimers are bound to O L 1, O L 2, O R 1, and O R 2 a loop is induced in the DNA, allowing these dimers to bind together to form an octamer. This is a phenomenon called  long-range cooperativity . Upon formation of the octamer, cI dimers may cooperatively bind to O L 3 and O R 3, repressing transcription of cI. This  autonegative  regulation ensures a stable minimum concentration of the repressor molecule and, should SOS signals arise, allows for more efficient prophage induction. [ 25 ]


An important distinction here is that between the two decisions; lysogeny and lysis on infection, and continuing lysogeny or lysis from a prophage. The latter is determined solely by the activation of RecA in the SOS response of the cell, as detailed in the section on induction. The former will also be affected by this; a cell undergoing an SOS response will always be lysed, as no cI protein will be allowed to build up. However, the initial lytic/lysogenic decision on infection is also dependent on the cII and cIII proteins.


In cells with sufficient nutrients, protease activity is high, which breaks down cII. This leads to the lytic lifestyle. In cells with limited nutrients, protease activity is low, making cII stable. This leads to the lysogenic lifestyle. cIII appears to stabilize cII, both directly and by acting as a competitive inhibitor to the relevant proteases. This means that a cell "in trouble", i.e. lacking in nutrients and in a more dormant state, is more likely to lysogenise. This would be selected for because the phage can now lie dormant in the bacterium until it falls on better times, and so the phage can create more copies of itself with the additional resources available and with the more likely proximity of further infectable cells.


A full biophysical model for lambda's lysis-lysogeny decision remains to be developed. Computer modeling and simulation suggest that random processes during infection drive the selection of lysis or lysogeny within individual cells. [ 26 ]   However, recent experiments suggest that physical differences among cells, that exist prior to infection, predetermine whether a cell will lyse or become a lysogen. [ 27 ]


Lambda phage has been used heavily as a  model organism  and has been an excellent tool first in  microbial genetics , and then later in  molecular genetics . [ 28 ]  Some of its uses include its application as a vector for the cloning of  recombinant DNA ; the use of its site-specific recombinase (int) for the shuffling of cloned DNAs by the  gateway method ; [ 29 ]  and the application of its Red  operon , including the proteins Red alpha (also called 'exo'), beta and gamma in the DNA engineering method called  recombineering . The 48 kb DNA fragment of lambda phage is not essential for productive infection and can be replaced by foreign DNA, [ 30 ]  which can then be replicated by the phage. Lambda phage will enter bacteria more easily than plasmids, making it a useful vector that can either destroy or become part of the host's DNA. [ 31 ]  Lambda phage can also be manipulated and used as an anti-cancer vaccine that targets human  aspartyl (asparaginyl) β-hydroxylase  (ASPH, HAAH), which has been shown to be beneficial in cases of hepatocellular carcinoma in mice. [ 32 ]  Lambda phage has also been of major importance in the study of  specialized transduction . [ 33 ]


Loop-mediated isothermal amplification  ( LAMP ) is a single-tube technique for the amplification of  DNA [ 2 ]  for diagnostic purposes and a low-cost alternative to detect certain diseases. [ 3 ]  LAMP is an  isothermal nucleic acid amplification  technique. In contrast to the  polymerase chain reaction  (PCR) technology, in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a  thermal cycler . LAMP was invented in 1998 by  Eiken Chemical Company  in Tokyo. [ 1 ]   Reverse transcription loop-mediated isothermal amplification  (RT-LAMP) combines LAMP with a  reverse transcription  step to allow the detection of RNA.


In LAMP, the target sequence is amplified at a constant temperature of 60–65 °C (140–149 °F) using either two or three sets of  primers  and a  polymerase  like  Bst Klenow fragment  with high strand displacement activity in addition to a replication activity. Typically, four different primers are used to amplify six distinct regions on the target gene, which increases specificity. An additional pair of "loop primers" can further accelerate the reaction. [ 4 ]  The amount of DNA produced in LAMP is considerably higher than  PCR -based amplification. [ 1 ]  Primer design could be performed using several programs, such as  PrimerExplorer ,  MorphoCatcher , [ 5 ]  and  NEB LAMP Primer Design Tool . For the screening of conservative and species-specific nucleotide polymorphisms, in most diagnostics applications a combination of PrimerExplorer and MorphoCatcher is very useful, because it allows for the localization of species-specific nucleotides at 3'-ends of primers to enhance the specificity of reactions.


The amplification product can be detected via  photometry , measuring the turbidity caused by magnesium  pyrophosphate  precipitate in solution as a byproduct of amplification. [ 7 ]  This allows easy visualization by the naked eye or via simple photometric detection approaches for small volumes. The reaction can be followed in real-time either by measuring the turbidity [ 8 ]  or by fluorescence using intercalating dyes such as SYTO 9. [ 9 ]


Dyes, such as  SYBR green , can be used to create a visible color change that can be seen with the naked eye without the need for expensive equipment, or for a response that can more accurately be measured by instrumentation. Dye molecules intercalate or directly label the DNA, and in turn can be correlated with the number of copies initially present. Hence, LAMP can also be quantitative. In-tube detection of LAMP DNA amplification is possible using manganese loaded  calcein  which starts fluorescing upon complexation of manganese by pyrophosphate during in vitro DNA synthesis. [ 10 ]  Another method for visual detection of the LAMP amplicons by the unaided eye was based on their ability to hybridize with complementary gold nanoparticle-bound (AuNP) single-stranded DNA (ssDNA) and thus prevent the normal red to purple-blue color change that would otherwise occur during salt-induced aggregation of the gold particles. So, a LAMP method combined with amplicon detection by AuNP can have advantages over other methods in terms of reduced assay time, amplicon confirmation by hybridization and use of simpler equipment (i.e., no need for a thermocycler, electrophoresis equipment or a UV trans-illuminator). [ 11 ] [ 12 ]


pH-dependent dye indicators such as  Phenol Red  induce a color change from pink to yellow when the pH value of the reaction decreases upon DNA amplification. [ 13 ]  Due to its pronounced color change, this is the most commonly used readout for RT-LAMP assays. [ 13 ]  However, the pH-change dependent readout requires a weakly buffered reaction solution, which poses a great challenge when using crude sample inputs with variable pH. [ 13 ]  A second colorimetric assay utilizes  metal ion indicators  such as  hydroxynaphthol blue  (HNB), which changes color from purple to blue upon a drop in free Mg 2+  ions, which form a Mg- pyrophosphate  precipitate upon DNA amplification. [ 13 ]


LAMP is a relatively new DNA amplification technique, which due to its simplicity, ruggedness, and low cost could provide major advantages.
LAMP has the potential to be used as a simple screening assay in the field or at the point of care by clinicians. [ 14 ]
Because LAMP is isothermal, which eradicates the need for expensive thermocyclers used in conventional PCR, it may be a particularly useful method for infectious disease diagnosis in low and middle income countries. [ 15 ]  LAMP is widely being studied for detecting infectious diseases such as filariasis, [ 16 ]  Zika Virus, [ 17 ]  tuberculosis, [ 18 ]  malaria, [ 19 ] [ 20 ] [ 21 ]  sleeping sickness, [ 22 ]  and  SARS-CoV-2 . [ 23 ] [ 24 ]  In developing regions, it has yet to be extensively validated for other common pathogens. [ 14 ]


LAMP has been observed to be less sensitive (more resistant) than PCR to inhibitors in complex samples such as blood, likely due to use of a different DNA polymerase (typically  Bst  –  Bacillus stearothermophilus  – DNA polymerase rather than  Taq  polymerase as in PCR). Several reports describe successful detection of pathogens from minimally processed samples such as heat-treated blood, [ 25 ] [ 26 ]  or in presence of clinical sample matrices. [ 27 ]   This feature of LAMP may be useful in low-resource or field settings where a conventional DNA or RNA extraction prior to diagnostic testing may be impractical.


LAMP has also been used in helping identify body fluids. With its simplicity, researchers are able to test one or more samples with little hands on time which is helping cut down the time needed to get results. Researchers have also been able to add factors to make identification even more simple including metal-indicator dye and phenol red to be able to use a smartphone and the naked eye respectively to analyze the results. [ 28 ] [ 29 ] [ 30 ]


LAMP is less versatile than PCR, the most well-established nucleic acid amplification technique. LAMP is useful primarily as a diagnostic or detection technique, but is not useful for cloning or many other molecular biology applications enabled by PCR. Because LAMP uses 4 (or 6) primers targeting 6 (or 8) regions within a fairly small segment of the genome, and because primer design is subject to numerous constraints, it is difficult to design primer sets for LAMP "by eye". Free, open-source [ 31 ]   or commercial software packages are generally used to assist with LAMP primer design, although the primer design constraints mean there is less freedom to choose the target site than with PCR.


In a diagnostic application, this must be balanced against the need to choose an appropriate target (e.g., a conserved site in a highly variable viral genome, or a target that is specific for a particular strain of pathogen). Multiple degenerated sequences may be required to cover the different variant strains of the same species. A consequence of having such a cocktail of primers can be non-specific amplification in the late amplification. [ citation needed ]


Multiplexing approaches for LAMP are less developed than for PCR. The larger number of primers per target in LAMP increases the likelihood of primer-primer interactions for multiplexed target sets. The product of LAMP is a series of concatemers of the target region, giving rise to a characteristic "ladder" or banding pattern on a gel, rather than a single band as with PCR. Although this is not a problem when detecting single targets with LAMP, "traditional" (endpoint) multiplex PCR applications wherein identity of a target is confirmed by size of a band on a gel are not feasible with LAMP. Multiplexing in LAMP has been achieved by choosing a target region with a restriction site, and digesting prior to running on a gel, such that each product gives rise to a distinct size of fragment, [ 32 ]  although this approach adds complexity to the experimental design and protocol.


The use of a strand-displacing DNA polymerase in LAMP also precludes the use of hydrolysis probes, e.g.  TaqMan  probes, which rely upon the 5'-3'  exonuclease  activity of  Taq  polymerase. An alternative real-time multiplexing approach based on fluorescence quenchers has been reported. [ 33 ]


SYBR green dye may be added to view LAMP in real-time. However, in the late amplification, primer-dimer amplification may contribute to a false positive signal. The use of  inorganic pyrophosphatase  in a SYBR reaction mix allows the use of melt analysis to distinguish correct amplification  [ 34 ]


Although different mitigation strategies have been proposed for false-positive results in assays based on this method, nonspecific amplification due to various factors including the absence of temperature gating mechanisms is one of the major limitations of Loop-mediated isothermal amplification. [ 35 ] [ 36 ]


Lastly, because LAMP requires maintained, elevated incubation temperatures (60–65 °C), some sort of heating mechanism, thermostat, and/or insulator is required (though not necessarily a thermal cycler). This requirement makes LAMP less ideally suited for in the field, point-of-care diagnostics which would ideally function at ambient temperature. [ citation needed ]


RNase hybridization-assisted amplification (RHAM) integrates LAMP with  RNase HII -mediated fluorescent reporting. This method employs a conventional LAMP primer set to exponentially amplify the target sequence, followed by the hybridization of a ribonucleotide-containing fluorescent probe to the amplification product. RNase HII then cleaves the probe, releasing a fluorescent signal that can be detected. [ 37 ] [ 38 ]


Methylthioninium chloride , commonly called  methylene blue , is a salt used as a  dye  and as a medication. As a medication, it is mainly used to treat  methemoglobinemia  by  chemically reducing  the  ferric iron  in  hemoglobin  to  ferrous iron . [ 6 ] [ 2 ]  Specifically, it is used to treat methemoglobin levels that are greater than 30% or in which there are symptoms despite  oxygen therapy . [ 2 ]  It has previously been used for treating  cyanide poisoning  and  urinary tract infections , but this use is no longer recommended. [ 6 ]


Methylene blue is typically given by  injection into a vein . [ 6 ]  Common side effects include  headache ,  nausea , and  vomiting . While use during  pregnancy  may harm the fetus, not using it in methemoglobinemia is likely more dangerous. [ 6 ] [ 2 ]


Methylene blue was first prepared in 1876, by  Heinrich Caro . [ 9 ]  It is on the  World Health Organization's List of Essential Medicines . [ 10 ]


Methylene blue is employed as a medication for the treatment of  methemoglobinemia , which can arise from ingestion of certain pharmaceuticals, toxins, or  broad beans  in those susceptible. [ 11 ]   Normally, through the  NADH - or  NADPH -dependent  methemoglobin reductase  enzymes, methemoglobin is reduced back to hemoglobin. When large amounts of methemoglobin occur secondary to toxins, methemoglobin reductases are overwhelmed. Methylene blue, when injected intravenously as an antidote, is itself first reduced to leucomethylene blue, which then reduces the  heme  group from  methemoglobin  to  hemoglobin . Methylene blue can reduce the half life of methemoglobin from hours to minutes. [ 12 ]   At high doses, however, methylene blue actually induces methemoglobinemia, reversing this pathway. [ 12 ]


Since its reduction potential is similar to that of oxygen and can be reduced by components of the  electron transport chain , large doses of methylene blue are sometimes used as an antidote to  potassium cyanide  poisoning, a method first successfully tested in 1933 by  Matilda Moldenhauer Brooks  in San Francisco, [ 15 ]  although first demonstrated by Bo Sahlin of  Lund University , in 1926. [ 15 ] [ 16 ]


Methylene blue is used in  endoscopic   polypectomy  as an adjunct to  saline  or  epinephrine , and is used for injection into the submucosa around the polyp to be removed. This allows the submucosal tissue plane to be identified after the polyp is removed, which is useful in determining if more tissue needs to be removed, or if there has been a high risk for perforation. Methylene blue is also used as a dye in  chromoendoscopy , and is sprayed onto the mucosa of the  gastrointestinal tract  in order to identify  dysplasia , or pre-cancerous lesions. Intravenously injected methylene blue is readily released into the urine and thus can be used to test the  urinary tract  for leaks or  fistulas . [ citation needed ]


In surgeries such as  sentinel lymph node  dissections, methylene blue can be used to visually trace the lymphatic drainage of tested tissues. Similarly, methylene blue is added to  bone cement  in orthopedic operations to provide easy discrimination between native bone and cement. Additionally, methylene blue accelerates the hardening of bone cement, increasing the speed at which bone cement can be effectively applied. Methylene blue is used as an aid to visualisation/orientation in a number of medical devices, including a  Surgical sealant film , TissuePatch. In  fistulas  and  pilonidal sinuses  it is used to identify the tract for complete excision. [ citation needed ]  It can also be used during gastrointestinal surgeries (such as  bowel resection  or  gastric bypass ) to test for leaks. [ citation needed ]


It is sometimes used in  cytopathology , in mixtures including  Wright-Giemsa  and  Diff-Quik . It confers a blue color to both nuclei and cytoplasm, and makes the nuclei more visible. [ 17 ]  When methylene blue is "polychromed" (oxidized in solution or "ripened" by fungal metabolism, [ 18 ]  as originally noted in the thesis of  Dr. D. L. Romanowsky  in the 1890s), it gets serially demethylated and forms all the tri-, di-, mono- and non-methyl intermediates, which are  Azure B ,  Azure A ,  Azure C , and  thionine , respectively. [ 19 ]  This is the basis of the basophilic part of the spectrum of  Romanowski-Giemsa  effect. If only synthetic Azure B and  Eosin Y  is used, it may serve as a standardized  Giemsa stain ; but, without methylene blue, the normal neutrophilic granules tend to overstain and look like toxic granules. On the other hand, if methylene blue is used it might help to give the normal look of neutrophil granules and may also enhance the staining of nucleoli and polychromatophilic RBCs (reticulocytes). [ 20 ]


A traditional application of methylene blue is the intravital or supravital staining of nerve fibers, an effect first described by  Paul Ehrlich  in 1887. [ 21 ]  A dilute solution of the dye is either injected into tissue or applied to small freshly removed pieces. The selective blue coloration develops with exposure to air (oxygen) and can be fixed by immersion of the stained specimen in an aqueous solution of  ammonium molybdate . Vital methylene blue was formerly much used for examining the innervation of muscle, skin and internal organs. [ 22 ] [ 23 ] [ 24 ]  The mechanism of selective dye uptake is incompletely understood; vital staining of nerve fibers in skin is prevented by  ouabain , a drug that inhibits the Na/K-ATPase of cell membranes. [ 25 ]


Methylene blue has been used as a  placebo ; physicians would tell their patients to expect their urine to change color and view this as a sign that their condition had improved. [ 26 ]  This same side effect makes methylene blue difficult to use in traditional placebo-controlled  clinical studies , including those testing for its efficacy as a treatment. [ 27 ]


Isobutyl nitrite  is one of the compounds used as  poppers , an  inhalant  drug that induces a brief  euphoria .


Isobutyl nitrite is known to cause  methemoglobinemia . [ 28 ]  Severe methemoglobinemia may be treated with methylene blue. [ 29 ]


Another use of methylene blue is to treat  ifosfamide   neurotoxicity . Methylene blue was first reported for treatment and  prophylaxis  of ifosfamide neuropsychiatric toxicity in 1994. A toxic metabolite of ifosfamide,  chloroacetaldehyde  (CAA), disrupts the mitochondrial  respiratory chain , leading to an accumulation of  nicotinamide adenine dinucleotide  hydrogen (NADH). Methylene blue acts as an alternative electron acceptor, and reverses the NADH inhibition of hepatic  gluconeogenesis  while also inhibiting the transformation of chloroethylamine into chloroacetaldehyde, and inhibits multiple amine oxidase activities, preventing the formation of CAA. [ 30 ]   The dosing of methylene blue for treatment of ifosfamide neurotoxicity varies, depending upon its use simultaneously as an adjuvant in ifosfamide infusion, versus its use to reverse psychiatric symptoms that manifest after completion of an ifosfamide infusion. Reports suggest that methylene blue up to six doses a day have resulted in improvement of symptoms within 10 minutes to several days. [ 31 ]  Alternatively, it has been suggested that intravenous methylene blue every six hours for prophylaxis during ifosfamide treatment in people with history of ifosfamide neuropsychiatric toxicity. [ 32 ]   Prophylactic administration of methylene blue the day before initiation of ifosfamide, and three times daily during ifosfamide chemotherapy has been recommended to lower the occurrence of ifosfamide neurotoxicity. [ 33 ]


It has also been used in  septic shock  and  anaphylaxis . [ 34 ] [ 35 ]


Methylene blue consistently increases blood pressure in people with  vasoplegic syndrome  (redistributive shock), but has not been shown to improve delivery of oxygen to tissues or to decrease mortality. [ 36 ] [ 37 ] [ 38 ] [ 39 ]


Methylene blue has been used in  calcium channel blocker  toxicity as a rescue therapy for distributive shock unresponsive to first line agents. Evidence for its use in this circumstance is very poor and limited to a handful of case reports. [ 40 ] [ 41 ] [ 42 ] [ 43 ] [ 44 ] [ 45 ] [ 46 ] [ 47 ] [ 48 ] [ excessive citations ]


Methylene blue is a  monoamine oxidase inhibitor  (MAOI) [ 51 ]  and, if infused intravenously at doses exceeding 5 mg/kg, may result in  serotonin syndrome  if combined with any  selective serotonin reuptake inhibitors  (SSRIs) or other serotonergic drugs (e.g.,  duloxetine ,  sibutramine ,  venlafaxine ,  clomipramine ,  imipramine ). [ 52 ]


It causes  hemolytic anemia  in carriers of the  G6PD  ( favism ) enzymatic deficiency. [ 53 ]


Methylene blue is a formal derivative of  phenothiazine . It is a dark green powder that yields a blue solution in  water . The hydrated form has 3 molecules of water per unit of methylene blue.


This compound is prepared by oxidation of 4-aminodimethylaniline in the presence of  sodium thiosulfate  to give the quinonediiminothiosulfonic acid, reaction with dimethylaniline, oxidation to the indamine, and cyclization to give the thiazine: [ 54 ]


A green electrochemical procedure, using only  dimethyl-4-phenylenediamine  and sulfide ions has been proposed. [ 55 ]


The maximum absorption of light is near 670 nm. The specifics of absorption depend on a number of factors, including  protonation ,  adsorption  to other materials, and  metachromasy  - the formation of  dimers  and higher-order aggregates depending on concentration and other interactions: [ 56 ]


Methylene blue is widely used as a  redox indicator  in  analytical chemistry . [ 57 ] [ 58 ] [ 59 ]  Solutions of this substance are blue when in an oxidizing environment, but will turn colorless if exposed to a reducing agent. [ 60 ] [ 58 ]  The redox properties can be seen in a classical demonstration of  chemical kinetics  in general chemistry, the " blue bottle " experiment. Typically, a solution is made of  glucose  (dextrose), methylene blue, and  sodium hydroxide . Upon shaking the bottle,  oxygen  oxidizes methylene blue, and the solution turns blue. The dextrose will gradually reduce the methylene blue to its colorless, reduced form. Hence, when the dissolved dextrose is entirely consumed, the solution will turn blue again. The redox midpoint potential E 0 ' is +0.01 V. [ 61 ]


Methylene blue is also a  photosensitizer  used to create  singlet oxygen  when exposed to both oxygen and light. It is used in this regard to make organic  peroxides  by a  Diels-Alder  reaction which is  spin forbidden  with normal atmospheric  triplet oxygen . [ citation needed ]


The formation of methylene blue after the reaction of  hydrogen sulfide  with  dimethyl-p-phenylenediamine  and  iron(III)  at pH 0.4 – 0.7 is used to determine by  photometric measurements   sulfide  concentration in the range 0.020 to 1.50 mg/L (20 ppb to 1.5 ppm). [ 62 ]  The test is very sensitive and the blue coloration developing upon contact of the reagents with dissolved H 2 S is stable for 60 min. Ready-to-use kits such as the  Spectroquant   sulfide  test [ 63 ]  facilitate routine analyses. The methylene blue sulfide test is a convenient method often used in soil microbiology to quickly detect in water the metabolic activity of  sulfate reducing bacteria  (SRB). In this colorimetric test, methylene blue is a product formed by the reaction and not a reagent added to the system. [ 62 ]


The addition of a strong  reducing agent , such as  ascorbic acid , to a sulfide-containing solution is sometimes used to prevent sulfide oxidation from atmospheric oxygen. Although it is certainly a sound precaution for the determination of sulfide with an  ion selective electrode , it might however hamper the development of the blue color if the freshly formed methylene blue is also reduced, as described here above in the paragraph on redox indicator. [ 58 ]


Methylene blue is a dye behaving as a  redox indicator  that is commonly used in the  food industry  to test the freshness of  milk  and  dairy  products. [ 6 ] [ 64 ]  A few drops of methylene blue solution added to a sample of milk should remain blue (oxidized form in the presence of enough dissolved  O 2 ), otherwise (discoloration caused by the reduction of methylene blue into its colorless reduced form) the dissolved  O 2  concentration in the milk sample is low indicating that the milk is not fresh (already abiotically oxidized by  O 2  whose concentration in solution decreases) or could be contaminated by  bacteria  also consuming the atmospheric  O 2  dissolved in the milk. [ 64 ]  In other words,  aerobic  conditions should prevail in fresh milk and methylene blue is simply used as an indicator of the dissolved oxygen remaining in the milk. [ 60 ]


The adsorption of methylene blue serves as an indicator defining the adsorptive capacity of granular  activated carbon  in water filters. Adsorption of methylene blue is very similar to adsorption of pesticides from water, this quality makes methylene blue serve as a good predictor for filtration qualities of carbon. It is as well a quick method of comparing different batches of activated carbon of the same quality.
A  color reaction  in an acidified, aqueous methylene blue solution containing  chloroform  can detect  anionic surfactants  in a water sample. Such a test is known as an  MBAS assay  (methylene blue active substances assay).


The MBAS assay cannot distinguish between specific surfactants, however. Some examples of anionic surfactants are  carboxylates ,  phosphates ,  sulfates , and  sulfonates . [ citation needed ]


The methylene blue value is defined as the number of milliliter's standard methylene value solution decolorized 0.1 g of  activated carbon  (dry basis). [ 65 ]
Methylene blue value reflects the amount of clay minerals in  aggregate  samples. [ 66 ]  In   materials science , methylene blue solution is successively added to fine aggregate which is being agitated in water.  The presence of free dye solution can be checked with stain test on a filter paper. [ 67 ]


In biology, methylene blue is used as a  dye  for a number of different staining procedures, such as  Wright's stain  and  Jenner's stain . Since it is a temporary staining technique, methylene blue can also be used to examine  RNA  or  DNA  under the  microscope  or in a gel: as an example, a solution of methylene blue can be used to stain RNA on hybridization membranes in  northern blotting  to verify the amount of nucleic acid present. While methylene blue is not as sensitive as  ethidium bromide , it is less toxic and it does not  intercalate  in nucleic acid chains, thus avoiding interference with nucleic acid retention on hybridization membranes or with the hybridization process itself. [ citation needed ]


It can also be used as an indicator to determine whether eukaryotic cells such as yeast are alive or dead. The methylene blue is reduced in viable cells, leaving them unstained. However dead cells are unable to reduce the oxidized methylene blue and the cells are stained blue. Methylene blue can interfere with the respiration of the yeast as it picks up hydrogen ions made during the process. [ citation needed ]


Methylene blue is used in  aquaculture  and by tropical fish hobbyists as a treatment for fungal infections. It can also be effective in treating fish infected with  ich  although a combination of  malachite green  and  formaldehyde  is far more effective against the parasitic  protozoa   Ichthyophthirius multifiliis . It is usually used to protect newly laid fish eggs from being infected by fungus or bacteria. This is useful when the hobbyist wants to artificially hatch the fish eggs.
Methylene blue is also very effective when used as part of a "medicated fish bath" for treatment of ammonia, nitrite, and cyanide poisoning as well as for topical and internal treatment of injured or sick fish as a "first response". [ 68 ]


Methylene blue has been described as "the first fully synthetic drug used in medicine." Methylene blue was first prepared in 1876 by German chemist  Heinrich Caro . [ 69 ]


Its use in the treatment of malaria was pioneered by  Paul Guttmann  and  Paul Ehrlich  in 1891. During this period before the first World War, researchers like Ehrlich believed that drugs and dyes worked in the same way, by preferentially staining pathogens and possibly harming them. Changing the  cell membrane  of pathogens is in fact how various drugs work, so the theory was partially correct although far from complete. Methylene blue continued to be used in the second World War, where it was not well liked by soldiers, who observed, "Even at the loo, we see, we pee, navy blue."  Antimalarial use of the drug has recently been revived. [ 70 ]  It was discovered to be an antidote to  carbon monoxide poisoning  and  cyanide poisoning  in 1933 by  Matilda Brooks . [ 71 ]


Minimum information about a microarray experiment  ( MIAME ) is a standard created by the  FGED Society  for reporting  microarray  experiments. [ 1 ]


MIAME is intended to specify all the information necessary to interpret the results of the  experiment  unambiguously and to potentially reproduce the experiment. While the standard defines the content required for compliant reports, it does not specify the format in which this data should be presented. MIAME describes the minimum information required to ensure that microarray data can be easily interpreted and that results derived from its analysis can be independently verified. [ 2 ]  There are a number of  file formats  used to represent this data, as well as both public and subscription-based repositories for such experiments. [ 2 ]  Additionally, software exists to aid the preparation of MIAME-compliant reports.


MIAME revolves around six key components:  raw data ,  normalized data , sample annotations,  experimental design , array annotations, and data protocols.


Molecular beacons , or  molecular beacon probes , are  oligonucleotide   hybridization probes  that can report the presence of specific  nucleic acids  in homogenous solutions. Molecular beacons are  hairpin -shaped molecules with an internally  quenched   fluorophore  whose  fluorescence  is restored when they bind to a target nucleic acid sequence. This is a novel non- radioactive  method for detecting specific sequences of nucleic acids. They are useful in situations where it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes.


A typical molecular beacon probe is 25  nucleotides  long. [ citation needed ]  The middle 15 nucleotides are complementary to the target  DNA  or  RNA  and do not base pair with one another, while the five nucleotides at each terminus are complementary to each other rather than to the target DNA. A typical molecular beacon structure can be divided in 4 parts: 1) loop, an 18–30 base pair region of the molecular beacon that is complementary to the target sequence; 2) stem formed by the attachment to both termini of the loop of two short (5 to 7 nucleotide residues) oligonucleotides that are complementary to each other; 3) 5' fluorophore at the 5' end of the molecular beacon, a fluorescent dye is covalently attached; 4) 3' quencher (non fluorescent) dye that is covalently attached to the 3' end of the molecular beacon. When the beacon is in closed loop shape, the quencher resides in proximity to the fluorophore, which results in quenching the fluorescent emission of the latter.


If the nucleic acid to be detected is complementary to the strand in the loop, the event of  hybridization  occurs. The duplex formed between the nucleic acid and the loop is more stable than that of the stem because the former duplex involves more base pairs. This causes the separation of the stem and hence of the fluorophore and the quencher. Once the fluorophore is no longer next to the quencher, illumination of the hybrid with light results in the fluorescent emission. The presence of the emission reports that the event of hybridization has occurred and hence the target nucleic acid sequence is present in the test sample.


Fluorogenic signaling oligonucleotide probes were reported for use to detect and isolate cells expressing one or more desired genes, including the production of multigene stable cell lines expressing heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABAA) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs). [ 1 ]


Molecular beacons are  synthetic   oligonucleotides  whose preparation is well documented. In addition to the conventional set of  nucleoside phosphoramidites , the synthesis also requires a solid support derivatized with a quencher and a phosphoramidite building block designed for the attachment of a protected fluorescent dye.


The first use of the term molecular beacons, synthesis and demonstration of function was in 1996. [ 2 ]


Molecular cloning  is a set of experimental methods in  molecular biology  that are used to assemble  recombinant DNA  molecules and to direct their  replication  within  host organisms . [ 1 ]  The use of the word  cloning  refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living  host  for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine. [ 2 ]


In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with  vector DNA  to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of  E. coli  bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are  transgenic  or genetically modified microorganisms ( GMOs ). [ 3 ]  This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA molecule. This single cell can then be expanded exponentially to generate a large number of bacteria, each of which contains copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as "clones". Strictly speaking,  recombinant DNA  refers to DNA molecules, while  molecular cloning  refers to the experimental methods used to assemble them. The idea arose that different DNA sequences could be inserted into a  plasmid  and that these foreign sequences would be carried into bacteria and digested as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes. [ 4 ]


Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centromeres and telomeres. There is also a lower chance of success when inserting large-sized DNA sequences. Inserts larger than 10kbp have very limited success, but bacteriophages such as  bacteriophage λ  can be modified to successfully insert a sequence up to 40 kbp. [ 5 ]


Prior to the 1970s, the understanding of genetics and molecular biology was severely hampered by an inability to isolate and study individual genes from complex organisms. This changed dramatically with the advent of molecular cloning methods. Microbiologists, seeking to understand the molecular mechanisms through which bacteria restricted the growth of bacteriophage, isolated  restriction endonucleases , enzymes that could cleave DNA molecules only when specific DNA sequences were encountered. [ 6 ]  They showed that restriction enzymes cleaved chromosome-length DNA molecules at specific locations, and that specific sections of the larger molecule could be purified by size fractionation.  Using a second enzyme,  DNA ligase , fragments generated by restriction enzymes could be joined in new combinations, termed  recombinant DNA . By recombining DNA segments of interest with vector DNA, such as bacteriophage or plasmids, which naturally replicate inside bacteria, large quantities of purified recombinant DNA molecules could be produced in bacterial cultures. The first recombinant DNA molecules were generated and studied in 1972. [ 7 ] [ 8 ]


Molecular cloning takes advantage of the fact that the chemical structure of  DNA  is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for  DNA replication , and the resulting  recombinant DNA  is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell's DNA in the  transgenic  organism.


Molecular cloning is similar to PCR in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an  in vitro  solution, free of living cells.


Before actual cloning experiments are performed in the lab, most cloning experiments are planned in a computer, using specialized software. Although the detailed planning of the cloning can be done in any text editor, together with online utilities for e.g. PCR primer design, dedicated software exist for the purpose. Software for the purpose include for example ApE  [1]  (open source), DNAStrider  [2]  (open source), Serial Cloner  [3]  (gratis), Collagene  [4]  (open source), and  SnapGene  (commercial). These programs allow to simulate  PCR reactions ,  restriction digests ,  ligations , etc., that is, all the steps described below.


In standard molecular cloning experiments, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into host organism, (6) Selection of organisms containing recombinant DNA, (7) Screening for clones with desired DNA inserts and biological properties.


Notably, the growing capacity and fidelity of DNA synthesis platforms allows for increasingly intricate designs in molecular engineering. These projects may include very long strands of novel DNA sequence and/or test entire libraries simultaneously, as opposed to of individual sequences. These shifts introduce complexity that require design to move away from the flat nucleotide-based representation and towards a higher level of abstraction. Examples of such tools are  GenoCAD , Teselagen  [5]  (free for academia) or GeneticConstructor  [6]  (free for academics).


Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium  E. coli  ( Escherichia coli ) and a  plasmid cloning vector .  E. coli  and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. [ 3 ]  If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a  bacterial artificial chromosome [ 10 ]  or  yeast artificial chromosome  vector is often chosen.


Specialized applications may call for specialized host-vector systems. For example, if the experimentalists wish to harvest a particular protein from the recombinant organism, then an  expression vector  is chosen that contains appropriate signals for transcription and translation in the desired host organism. Alternatively, if replication of the DNA in different species is desired (for example, transfer of DNA from bacteria to plants), then a multiple host range vector (also termed  shuttle vector ) may be selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by  subcloning  into a specialized vector.


Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility: [ 3 ]


The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with the ends of the foreign DNA (see  DNA end ). Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme or restriction endonuclease, for example  EcoRI  and this restriction enzyme was isolated from E.coli. [ 11 ]  Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule (so that the vector can only be cleaved at a single site) and are located within a gene (frequently  beta-galactosidase ) whose inactivation can be used to distinguish recombinant from non-recombinant organisms at a later step in the process. To improve the ratio of recombinant to non-recombinant organisms, the cleaved vector may be treated with an enzyme ( alkaline phosphatase ) that dephosphorylates the vector ends.  Vector molecules with dephosphorylated ends are unable to replicate, and replication can only be restored if foreign DNA is integrated into the cleavage site. [ 12 ]


For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest. Virtually any tissue source can be used (even tissues from  extinct animals ), [ 13 ]  as long as the DNA is not extensively degraded. The DNA is then purified using simple methods to remove contaminating proteins (extraction with phenol), RNA (ribonuclease) and smaller molecules (precipitation and/or chromatography).  Polymerase chain reaction  (PCR) methods are often used for amplification of specific DNA or RNA ( RT-PCR ) sequences prior to molecular cloning.


DNA for cloning experiments may also be obtained from RNA using reverse transcriptase ( complementary DNA  or cDNA cloning), or in the form of synthetic DNA ( artificial gene synthesis ). cDNA cloning is usually used to obtain clones representative of the mRNA population of the cells of interest, while synthetic DNA is used to obtain any precise sequence defined by the designer. Such a designed sequence may be required when moving genes across  genetic codes  (for example, from the mitochondria to the nucleus) [ 14 ]  or simply for increasing expression via  codon optimization . [ 15 ]


The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector. If necessary, short double-stranded segments of DNA ( linkers ) containing desired restriction sites may be added to create end structures that are compatible with the vector. [ 3 ] [ 12 ]


The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are simply mixed together at appropriate concentrations and exposed to an enzyme ( DNA ligase ) that covalently links the ends together. This joining reaction is often termed  ligation . The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism.


DNA ligase only recognizes and acts on the ends of linear DNA molecules, usually resulting in a complex mixture of DNA molecules with randomly joined ends. The desired products (vector DNA covalently linked to foreign DNA) will be present, but other sequences (e.g. foreign DNA linked to itself, vector DNA linked to itself and higher-order combinations of vector and foreign DNA) are also usually present. This complex mixture is sorted out in subsequent steps of the cloning process, after the DNA mixture is introduced into cells. [ 3 ] [ 12 ]


The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen (e.g.  transformation ,  transduction ,  transfection ,  electroporation ). [ 3 ] [ 12 ]


When microorganisms are able to take up and replicate DNA from their local environment, the process is termed  transformation , and cells that are in a physiological state such that they can take up DNA are said to be  competent . [ 16 ]  In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed  transfection . Both transformation and transfection usually require preparation of the cells through a special growth regime and chemical treatment process that will vary with the specific species and cell types that are used.


Electroporation  uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present). [ 17 ]  In contrast,  transduction  involves the packaging of DNA into virus-derived particles, and using these virus-like particles to introduce the encapsulated DNA into the cell through a process resembling viral infection. Although electroporation and transduction are highly specialized methods, they may be the most efficient methods to move DNA into cells.


Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive. [ 3 ] [ 12 ]


When bacterial cells are used as host organisms, the  selectable marker  is usually a gene that confers resistance to an  antibiotic  that would otherwise kill the cells, typically  ampicillin . Cells harboring the plasmid will survive when exposed to the antibiotic, while those that have failed to take up plasmid sequences will die. When mammalian cells (e.g. human or mouse cells) are used, a similar strategy is used, except that the marker gene (in this case typically encoded as part of the  kanMX  cassette) confers resistance to the antibiotic  Geneticin .


Modern bacterial cloning vectors (e.g.  pUC19  and later derivatives including the pGEM vectors) use the  blue-white screening system  to distinguish colonies (clones) of transgenic cells from those that contain the parental vector (i.e. vector DNA with no recombinant sequence inserted). In these vectors, foreign DNA is inserted into a sequence that encodes an essential part of  beta-galactosidase , an enzyme whose activity results in formation of a blue-colored colony on the culture medium that is used for this work. Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme so that colonies containing transformed DNA remain colorless (white). Therefore, experimentalists are easily able to identify and conduct further studies on transgenic bacterial clones, while ignoring those that do not contain recombinant DNA.


The total population of individual clones obtained in a molecular cloning experiment is often termed a  DNA library . Libraries may be highly complex (as when cloning complete genomic DNA from an organism) or relatively simple (as when moving a previously cloned DNA fragment into a different plasmid), but it is almost always necessary to examine a number of different clones to be sure that the desired DNA construct is obtained. This may be accomplished through a very wide range of experimental methods, including the use of  nucleic acid hybridizations ,  antibody probes ,  polymerase chain reaction ,  restriction fragment analysis  and/or  DNA sequencing . [ 3 ] [ 12 ]


Molecular cloning provides scientists with an essentially unlimited quantity of any individual DNA segments derived from any genome. This material can be used for a wide range of purposes, including those in both basic and applied biological science. A few of the more important applications are summarized here.


Molecular cloning has led directly to the elucidation of the complete DNA sequence of the genomes of a very large number of species and to an exploration of genetic diversity within individual species, work that has been done mostly by determining the DNA sequence of large numbers of randomly cloned fragments of the genome, and assembling the overlapping sequences.


At the level of individual genes, molecular clones are used to generate  probes  that are used for examining how genes are  expressed , and how that expression is related to other processes in biology, including the metabolic environment, extracellular signals, development, learning, senescence and cell death. Cloned genes can also provide tools to examine the biological function and importance of individual genes, by allowing investigators to  inactivate  the genes, or make more subtle mutations using regional mutagenesis or  site-directed mutagenesis . Genes cloned into expression vectors for  functional cloning  provide a means to screen for genes on the basis of the expressed protein's function.


Obtaining the molecular clone of a gene can lead to the development of organisms that produce the protein product of the cloned genes, termed a recombinant protein. In practice, it is frequently more difficult to develop an organism that produces an active form of the recombinant protein in desirable quantities than it is to clone the gene. This is because the molecular signals for gene expression are complex and variable, and because protein folding, stability and transport can be very challenging.


Many useful proteins are currently available as  recombinant products . These include--(1) medically useful proteins whose administration can correct a defective or poorly expressed gene (e.g. recombinant  factor VIII , a blood-clotting factor deficient in some forms of  hemophilia , [ 18 ]  and recombinant  insulin , used to treat some forms of  diabetes [ 19 ] ), (2) proteins that can be administered to assist in a life-threatening emergency (e.g.  tissue plasminogen activator , used to treat strokes [ 20 ] ), (3) recombinant subunit vaccines, in which a purified protein can be used to immunize patients against infectious diseases, without exposing them to the infectious agent itself (e.g.  hepatitis B vaccine [ 21 ] ), and (4) recombinant proteins as standard material for diagnostic laboratory tests.


Once characterized and manipulated to provide signals for appropriate expression, cloned genes may be inserted into organisms, generating transgenic organisms, also termed  genetically modified organisms  (GMOs). Although most GMOs are generated for purposes of basic biological research (see for example,  transgenic mouse ), a number of GMOs have been developed for commercial use, ranging from animals and plants that produce pharmaceuticals or other compounds ( pharming ),  herbicide-resistant crop plants , and fluorescent tropical fish ( GloFish ) for home entertainment. [ 1 ]


Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or acquired disease. Gene therapy can be broadly divided into two categories. The first is alteration of germ cells, that is, sperm or eggs, which results in a permanent genetic change for the whole organism and subsequent generations. This "germ line gene therapy" is considered by many to be unethical in human beings. [ 22 ]  The second type of gene therapy, "somatic cell gene therapy", is analogous to an organ transplant. In this case, one or more specific tissues are targeted by direct treatment or by removal of the tissue, addition of the therapeutic gene or genes in the laboratory, and return of the treated cells to the patient. Clinical trials of somatic cell gene therapy began in the late 1990s, mostly for the treatment of cancers and blood, liver, and lung disorders. [ 23 ]


Despite a great deal of publicity and promises, the history of human gene therapy has been characterized by relatively limited success. [ 23 ]  The effect of introducing a gene into cells often promotes only partial and/or transient relief from the symptoms of the disease being treated. Some gene therapy trial patients have suffered adverse consequences of the treatment itself, including deaths. In some cases, the adverse effects result from disruption of essential genes within the patient's genome by insertional inactivation. In others, viral vectors used for gene therapy have been contaminated with infectious virus. Nevertheless, gene therapy is still held to be a promising future area of medicine, and is an area where there is a significant level of research and development activity.


A  molecular-weight size marker , also referred to as a  protein  ladder ,  DNA  ladder , or  RNA  ladder , is a set of  standards  that are used to identify the  approximate  size of a  molecule  run on a  gel  during  electrophoresis , using the  principle  that  molecular weight  is inversely proportional to migration rate through a gel matrix. Therefore, when used in  gel electrophoresis , markers effectively provide a  logarithmic scale  by which to estimate the size of the other fragments (providing the fragment sizes of the marker are known).


Protein, DNA, and RNA markers with pre-determined fragment sizes and concentrations are commercially available. These can be run in either  agarose  or  polyacrylamide gels . The markers are loaded in lanes adjacent to sample lanes before the commencement of the run.


Although the concept of molecular-weight markers has been retained, techniques of development have varied throughout the years. New inventions of molecular-weight markers are distributed in kits specific to the marker's type.


An early problem in the development of markers was achieving high resolution throughout the entire length of the marker. [ 1 ]  Depending on the running conditions of gel electrophoresis, fragments may have been compressed, disrupting clarity. To address this issue, a kit for  Southern Blot  analysis was developed in 1990, providing the first marker to combine target DNA and probe DNA. This technique took advantage of logarithmic spacing, and could be used to identify target bands ranging over a length of 20,000  nucleotides . [ 2 ]


There are two common methods in which to construct a DNA molecular-weight size marker. [ 3 ]  One such method employs the technique of partial  ligation . [ 3 ]  DNA ligation is the process by which linear DNA pieces are connected to each other via  covalent bonds ; more specifically, these bonds are  phosphodiester bonds . [ 4 ]  Here, a 100bp duplex DNA piece is partially ligated. The consequence of this is that dimers of 200bp, trimers of 300bp, tetramers of 400bp, pentamers of 500bp, etc. will form. Additionally, a portion of the 100bp dsDNA will remain. As a result, a DNA "ladder" composed of DNA pieces of known  molecular mass  is created on the gel. [ 3 ]


The second method employs the use of  restriction enzymes  and a recognized DNA sequence. [ 3 ]  The DNA is  digested  by a particular restriction enzyme, resulting in DNA pieces of varying molecular masses. One of the advantages of this method is that more marker can readily be created simply by digesting more of the known DNA. [ 3 ]  On the other hand, the size of the DNA pieces are based on the sites where the restriction enzyme cuts. This makes it more difficult to control the size of the fragments in the marker. [ 5 ]


More recently, another method for constructing DNA molecular-weight size markers is being employed by laboratories. This strategy involves the use of  Polymerase Chain Reaction (PCR) . [ 5 ]  This is achieved one or two ways: 1) a DNA target is amplified at the same time via  primer  sets, or 2) different DNA targets are amplified independently via particular primers. [ 5 ]


As with experimental samples, the conditions of the gel can affect the molecular-weight size marker that runs alongside them. Factors such as  buffer , charge/ voltage , and  concentration  of gel can affect the  mobility  and/or appearance of your marker/ladder/standard. These elements need to be taken into consideration when selecting a marker and when analyzing the final results on a gel.


Previously, protein markers had been developed using a variety of whole proteins. The development of a kit including a molecular-weight size marker based on protein fragments began in 1993. This protein marker, composed of 49 different  amino acid  sequences, included  multidomain proteins , and allowed for the analysis of proteins cleaved at different sites. [ 9 ]


Current technique improvements in protein markers involve the use of auto-development. The first auto-developed regularly-weight protein marker was invented in 2012. [ 10 ]


Similar to DNA markers, these markers are typically composed of purified proteins whose molecular masses are already known. [ 3 ]  The list below outlines some of the proteins, as well as the molecular mass, that are commonly used when constructing a protein marker.


Molecular-weight size markers can be broken up into two categories: molecular weight markers vs. molecular ladder markers. [ 14 ]  Markers are either stained or unstained, and depending on the circumstance, one may be more appropriate than another. Molecular-weight size markers can also be biochemically altered. [ 15 ]  The conjugation with  biotin  is the most common. Molecular-weight size markers are most commonly used in  SDS-polyacrylamide gel electrophoresis  and  western blotting .
With all the different types and uses of molecular-weight size markers, it is important to choose the appropriate protein standard. Besides the most common use, as a way to calculate the molecular weight of the samples, other uses include allowing visual evidence of protein migration and transfer efficiency and are sometimes even used for positive control. [ 16 ]


As with DNA electrophoresis, conditions such as buffers, charge/voltage, and concentration should be taken into account when selecting a protein marker.


RNA ladders composed of RNA molecular-weight size markers were initially developed by using the synthetic circle method [ 21 ]  to produce different-sized markers. This technique was improved upon by inventor Eric T. Kool to use circular DNA  vectors  as a method for producing RNA molecular-weight size markers. As referred to as the rolling circle method, the improvements of this technique stems from its efficiency in synthesizing RNA  oligonucleotides . From the circular DNA template,  single-stranded RNA  varying in length from 4-1500 bp can be produced without the need for primers and by recycling  nucleotide triphosphate . DNA can also be synthesized from the circular template, adding to this technique's versatility. In comparison to  runoff transcription , the synthetic circle method produces RNA oligonucleotides without the runoff. In comparison to  PCR , the synthetic circle method produces RNA oligonucleotides without the need for polymerase nor a  thermal cycler . This method is also cost-efficient in its ability to synthesize grand amounts of product at a lower error rate than machine synthesizers. [ 21 ]


The RNA markers consist of RNA transcripts of various incrementing lengths. For example, the Lonza 0.5-9 kbp marker [ 22 ]  has bands marking 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 9  kilobase  pairs. Markers are dissolved in a storage buffer, such as  EDTA , and can have a  shelf life  of up to 2 years when stored at -80 °C. To use the marker, such as for northern blot analysis, it is first  thawed , and then stained so that it is detectable on a gel electrophoresis. One of the most common dyes used for markers is  ethidium bromide .


The range of a particular marker refers to variety of bands it can map. A "high" range refers to relatively large fragments (measured in kb) while a "low" range refers to markers that distinguish between small fragments (measured in bp). Some markers can even be described as "ultra-low range", [ 16 ]  but even more precise is the microRNA marker. A microRNA marker can be used to measure RNA fragments within a dozen nucleotides, such as the 17-25 nt microRNA marker. [ 23 ]


At equivalent molecular weights, RNA will migrate faster than DNA. However, both RNA and DNA have a negative  linear   slope  between their migration distance and  logarithmic  molecular weight. [ 24 ]  That is, samples of less weight are able to migrate a greater distance. This relationship is a consideration when choosing RNA or DNA markers as a standard.


When running RNA markers and RNA samples on a gel, it is important to prevent  nuclease  contamination, as RNA is very sensitive to  ribonuclease  (RNase) degradation through  catalysis . [ 25 ] [ 26 ]  Thus, all materials to be used in the procedure must be taken into consideration. Any glassware that is to come into contact with RNA should be pretreated with  diethylpyrocarbonate  (DEPC) and plastic materials should be disposable. [ 25 ]


One of the most common uses for molecular-weight size markers is in gel electrophoresis. The purpose of gel electrophoresis is to separate proteins by  physical  or  chemical properties , which include charge, molecular size, and  pH .< When separating based on size, the ideal method is SDS-PAGE or  polyacrylamide gel electrophoresis  and molecular-weight size markers are the appropriate standards to use.


Gels can vary in size. The number of samples to be run will determine the appropriate gel size. All gels are divided into lanes that run parallel through the gel. Each lane will contain a specific sample. Typically, molecular-weight size standards are placed in an outer lane. If a gel has a particularly high number of lanes, then multiple ladders may be placed across the gel for higher clarity.


Proteins and standards are  pipetted  on the gel in appropriate lanes.  Sodium dodecyl sulfate  (SDS) interacts with proteins,  denaturing  them, and giving them a negative charge. Since all proteins have the same charge-to-mass ratio, protein mobility through the gel will solely be based on molecular weight.  Once the electric field is turned on, protein migration will initiate. Upon completion, a detection mechanism such as western blotting can be used, which will reveal the presence of bands.  Each band represents a specific protein. The distance of travel is solely based on molecular weight; therefore, the molecular weight of each protein can be determined by comparing the distance of an unknown protein to the standard of known molecular weight. [ 27 ]


Many kinds of molecular-weight size markers exist, and each possess unique characteristics, lending to their involvement in a number of biological techniques. Selection of a molecular-weight size marker depends upon the marker type (DNA, RNA, or protein) and the length range it offers (e.g. 1kb). Before selecting a molecular-weight size marker, it is important to become familiar with these characteristics and properties. In a particular instance one type may be more appropriate than another. Although specific markers can vary between protocols for a given technique, this section will outline general markers and their roles.


The first type of molecular marker developed and run on gel electrophoresis were  allozymes . These markers are used for the detection of protein variation. The word "allozyme" (also known as "alloenzyme") comes from " allelic  variants of  enzymes ." [ 28 ]  When run on a gel, proteins are separated by size and charge. Although allozymes may seem dated when compared to the other markers available, they are still used today, mainly due to their low cost. One major downside is that since there is only a limited amount available, specificity an issue. [ 28 ]


Although allozymes can detect variations in DNA, it is by an indirect method and not very accurate. DNA-based markers were developed in the 1960s. [ 28 ]  These markers are much more effective at distinguishing between DNA variants. Today these are the most commonly used markers. DNA-based markers work by surveying nucleotides, which can serve a variety of functions, such as detecting differences in nucleotides or even quantifying the number of  mutations . [ 28 ]


The success of DNA based markers lead to the development of PCR. PCR ( polymerase chain reaction ) is a DNA  amplification  technique that can be applied to various types of fragments. Prior to this development, to amplify DNA, it had to be cloned or isolated. Shortly after the discovery of PCR came the idea of using PCR-based markers for gel electrophoresis. These type of markers are based on PCR  primers  and are categorized as DNA sequence  polymorphism . [ 28 ]


Although technically speaking, DNA sequence polymorphism has been going on since the use of RFLP in the 1960s, the analysis has changed significantly over the years. DNA sequence polymorphism uses older techniques like RFLP, but on a larger scale. Sequencing is much faster and more efficient. The analysis is automated, as it uses a technique known as shotgun sequencing. This high-throughput method is commonly used in population genetics. [ 28 ]


Carbohydrate  markers are employed in a technique known as  polysaccharide  analysis by carbohydrate gel electrophoresis (PACE), which is a measurable separation technique. [ 36 ]
It allows for the analysis of enzyme  hydrolysis  products. [ 36 ]  It has been used in applications such as characterizing enzymes involved in  hemicellulose  degradation, determining the structure of hemicellulose polysaccharides, and analysis of enzymatic cleavage of  cellulose  products. [ 36 ]


PACE depends on derivitization, which is the conversion of a chemical compound into a  derivative . [ 36 ] [ 37 ]  Here  monosaccharides ,  oligosaccharides , and polysaccharides are the compounds of interest. They are labeled at their reducing ends with a  fluorescent label  (i.e. a  fluorophore ). [ 36 ]  This derivitization with a fluorophore permits both separation on a gel under the desired circumstances and  fluorescence  imaging of the gel. In this case, a polyacrylamide gel is used. [ 36 ]


As with DNA, RNA, and protein electrophoresis, markers are run alongside the samples of interest in carbohydrate gel electrophoresis. [ 36 ]  The markers consist of oligosaccharides of known molecular weight. Like the samples of interest, the marker is also derivatized with a fluorophore (usually with 8-amino naphthalene -1,3,6- trisulfonic acid  (ANTS) or 2- aminoacridone ). [ 36 ]


A  Morpholino , also known as a  Morpholino oligomer  and as a  phosphorodiamidate Morpholino oligomer  ( PMO ), is a type of  oligomer   molecule  (colloquially, an  oligo ) used in  molecular biology  to modify  gene expression . Its  molecular structure  contains DNA bases attached to a backbone of methylenemorpholine rings linked through  phosphorodiamidate  groups. Morpholinos block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces of  ribonucleic acid  (RNA).  Morpholinos are used as research tools for  reverse genetics  by  knocking down  gene function.


This article discusses only the Morpholino antisense oligomers, which are  nucleic acid analogs . The word "Morpholino" can occur in other chemical names, referring to chemicals containing a six-membered  morpholine  ring. To help avoid confusion with other morpholine-containing molecules, when describing oligos "Morpholino" is often capitalized as a  trade name , but this usage is not consistent across scientific literature.  Morpholino oligos are sometimes referred to as PMO (for phosphorodiamidate morpholino oligomer), especially in medical literature.  Vivo-Morpholinos and PPMO are modified forms of Morpholinos with chemical groups covalently attached to facilitate entry into cells.


Gene knockdown  is achieved by reducing the expression of a particular gene in a cell. In the case of protein-coding genes, this usually leads to a reduction in the quantity of the corresponding protein in the cell. Knocking down gene expression is a method for learning about the function of a particular protein; in a similar manner, causing a specific  exon  to be spliced out of the RNA transcript encoding a protein can help to determine the function of the protein  moiety   encoded  by that exon or can sometimes knock down the protein activity altogether. These molecules have been applied to studies in several  model organisms , including  mice ,  zebrafish ,  frogs  and  sea urchins . [ 1 ]  Morpholinos can also modify the  splicing  of  pre-mRNA [ 2 ]  or inhibit the maturation and activity of miRNA. [ 3 ]   Techniques for targeting Morpholinos to RNAs and delivering Morpholinos into cells have recently been reviewed in a journal article [ 4 ]  and in book form. [ 5 ]


Morpholinos are in development as  pharmaceutical  therapeutics targeted against  pathogenic  organisms such as  bacteria [ 6 ]  or  viruses [ 7 ]  and  genetic diseases . [ 8 ]  A Morpholino-based drug  eteplirsen  from  Sarepta Therapeutics  received accelerated approval from the  US Food and Drug Administration  in September 2016 for the treatment of some mutations causing  Duchenne muscular dystrophy , [ 9 ]  although the approval process was mired in controversy. Other Morpholino-based drugs  golodirsen ,  viltolarsen , and  casimersen  (also for Duchenne muscular dystrophy) were approved by the FDA in 2019–2021. [ 10 ] [ 11 ] [ 12 ]


Morpholino oligos were conceived by Summerton ( Gene Tools ) at AntiVirals Inc. (now Sarepta Therapeutics) and originally developed in collaboration with Weller. [ 13 ]


Morpholinos are  synthetic   molecules  that are the product of a redesign of natural  nucleic acid  structure. [ 14 ]  Usually 25 bases in length, they bind to complementary sequences of RNA or single-stranded DNA by standard nucleic acid  base-pairing . In terms of structure, the difference between Morpholinos and  DNA  is that, while Morpholinos have standard nucleic acid bases, those bases are bound to methylene morpholine  rings linked through  phosphorodiamidate  groups instead of  phosphates . [ 14 ]  The figure compares the structures of the two strands depicted there, one of RNA and the other of a Morpholino. Replacement of  anionic  phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiological  pH  range, so Morpholinos in organisms or cells are uncharged molecules. The entire backbone of a Morpholino is made from these modified subunits.


Morpholinos do not trigger the degradation of their target RNA molecules, unlike many antisense structural types (e.g.,  phosphorothioates ,  siRNA ). Instead, Morpholinos act by "steric blocking", binding to a target sequence within an RNA, inhibiting molecules that might otherwise interact with the RNA. [ 15 ]  Morpholino oligos are often used to investigate the role of a specific mRNA transcript in an  embryo . Developmental biologists inject Morpholino oligos into eggs or embryos of  zebrafish , [ 16 ]  African clawed frog ( Xenopus ), [ 17 ]   sea urchin [ 18 ]  and killifish ( F. heteroclitus ) producing  morphant  embryos, or  electroporate  Morpholinos into  chick [ 19 ]  embryos at later development stages. With appropriate cytosolic delivery systems, Morpholinos are effective in  cell culture . [ 20 ] [ 21 ]  Vivo-Morpholinos, in which the oligo is covalently linked to a delivery  dendrimer , enter cells when administered systemically in adult animals or in tissue cultures. [ 22 ]


In  eukaryotic  organisms, pre-mRNA is  transcribed  in the nucleus,  introns  are  spliced  out, then the mature mRNA is exported from the  nucleus  to the  cytoplasm . The small subunit of the  ribosome  usually starts by binding at the 5' end of the mRNA and is joined there by various other  eukaryotic initiation factors , forming the initiation complex. The initiation complex scans along the mRNA strand until it reaches a  start codon , and then the large subunit of the ribosome attaches to the small subunit and  translation  of a  protein  begins. This entire process is referred to as gene expression; it is the process by which the information in a  gene , encoded as a sequence of bases in  DNA , is converted into the structure of a protein. A Morpholino can modify splicing, block translation, or block other functional sites on RNA depending on the Morpholino's base sequence.


Bound to the  5'-untranslated region  of messenger RNA (mRNA), Morpholinos can interfere with progression of the  ribosomal  initiation complex from the 5' cap to the start codon. This prevents  translation  of the coding region of the targeted  transcript  (called " knocking down "  gene expression ). This is useful experimentally when an investigator wishes to know the function of a particular protein; Morpholinos provide a convenient means of knocking down expression of the protein and learning how that knockdown changes the cells or organism. Some Morpholinos knock down expression so effectively that, after degradation of preexisting proteins, the targeted proteins become undetectable by  Western blot .


In 2016 a synthetic peptide-conjugated PMO (PPMO) was found to inhibit the expression of  New Delhi Metallo-beta-lactamase , an enzyme that many drug-resistant bacteria use to destroy carbapenems. [ 23 ] [ 24 ]


Morpholinos can interfere with  pre-mRNA  processing steps either by preventing splice-directing small nuclear ribonucleoproteins ( snRNP ) complexes from binding to their targets at the borders of introns on a strand of pre-mRNA, or by blocking the  nucleophilic   adenine  base and preventing it from forming the splice lariat structure, or by interfering with the binding of splice regulatory proteins such as splice silencers [ 25 ]  and  splice enhancers . [ 26 ]  Preventing the binding of snRNP  U1  (at the donor site) or  U2 / U5  (at the polypyrimidine moiety and acceptor site) can cause modified  splicing , commonly excluding  exons  from the mature mRNA. Targeting some splice targets results in intron inclusions, while activation of cryptic splice sites can lead to partial inclusions or exclusions. [ 27 ]  Targets of  U11 / U12  snRNPs can also be blocked. [ 28 ]  Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction ( RT-PCR ) and is seen as a band shift after  gel electrophoresis  of RT-PCR products. [ 2 ]


Morpholinos have been used to block  miRNA  activity [ 29 ] [ 30 ]  and maturation. [ 3 ]   Fluorescein -tagged Morpholinos combined with fluorescein-specific antibodies can be used as probes for  in-situ hybridization  to miRNAs. [ 31 ]  Morpholinos can block  ribozyme  activity. [ 32 ]   U2 and U12 snRNP functions have been inhibited by Morpholinos. [ 33 ]  Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational  frameshifts . [ 34 ]  Morpholinos can block RNA editing, [ 35 ]  poly(A) tailing [ 36 ]  and translocation sequences. [ 37 ]  Morpholino activities against this variety of targets suggest that Morpholinos can be used as a general-purpose tool for blocking interactions of proteins or nucleic acids with mRNA.


Morpholinos have become a standard knockdown tool in animal  embryonic  systems, which have a broader range of gene expression than adult  cells  and can be strongly affected by an off-target interaction. Following initial injections into frog or fish embryos at the single-cell or few-cell stages, Morpholino effects can be measured up to five days later, [ 38 ]  after most of the processes of  organogenesis  and  differentiation  are past, with observed  phenotypes  consistent with target-gene knockdown.  Control  oligos with irrelevant sequences usually produce no change in embryonic phenotype, evidence of the Morpholino oligo's sequence-specificity and lack of non-antisense effects. The dose required for a knockdown can be reduced by coinjection of several Morpholino oligos targeting the same mRNA, which is an effective strategy for reducing or eliminating dose-dependent off-target RNA interactions. [ 39 ]


mRNA rescue experiments can sometimes restore the  wild-type  phenotype to the embryos and provide evidence for the specificity of a Morpholino. In an mRNA rescue, a Morpholino is co-injected with an mRNA that codes for the morphlino's  protein. However, the rescue mRNA has a modified  5'-UTR  (untranslated region) so that the rescue mRNA contains no target for the Morpholino. The rescue mRNA's  coding region  encodes the protein of interest. Translation of the rescue mRNA replaces production of the protein that was knocked down by the Morpholino. Since the rescue mRNA would not affect phenotypic changes due to the Morpholino's off-target gene expression modulation, this return to wild-type phenotype is further evidence of Morpholino specificity. [ 38 ]   In some cases, ectopic expression of the rescue RNA makes recovery of the wild-type phenotype impossible.


In embryos, Morpholinos can be tested in null mutants to check for unexpected RNA interactions, then used in a wild-type embryo to reveal the acute knockdown phenotype. The knockdown phenotype is often more extreme than the mutant phenotype; in the mutant, effects of losing the null gene can be concealed by genetic compensation. [ 40 ]


Because of their completely unnatural backbones, Morpholinos are not recognized by cellular proteins.  Nucleases  do not degrade Morpholinos, [ 41 ]  nor are they degraded in serum or in cells. [ 42 ]


Up to 18% of Morpholinos appear to induce nontarget-related phenotypes including  cell death  in the  central nervous system  and  somite  tissues of zebrafish embryos. [ 43 ]   Most of these effects are due to activation of  p53 -mediated  apoptosis  and can be suppressed by co-injection of an anti-p53 Morpholino along with the experimental Morpholino. Moreover, the p53-mediated apoptotic effect of a Morpholino knockdown has been  phenocopied  using another antisense structural type, showing the p53-mediated apoptosis to be a consequence of the loss of the targeted protein and not a consequence of the knockdown oligo type. [ 44 ]   It appears that these effects are sequence-specific; as in most cases, if a Morpholino is associated with non-target effects, the 4-base mismatch Morpholino will not trigger these effects.


A cause for concern in the use of Morpholinos is the potential for "off-target" effects.  Whether an observed  morphant  phenotype is due to the intended knockdown or an interaction with an off-target RNA can often be addressed in embryos by running another experiment to confirm that the observed morphant phenotype results from the knockdown of the expected target. This can be done by recapitulating the morphant phenotype with a second, non-overlapping Morpholino targeting the same mRNA, [ 38 ]  by confirmation of the observed phenotypes by comparing with a mutant strain (though compensation will obscure a phenotype in some mutants), by testing the Morpholino in a null mutant background to detect additional phenotypic changes or by dominant-negative methods. As mentioned above, rescue of observed phenotypes by coinjecting a rescue mRNA is, when feasible, a reliable test of specificity of a Morpholino. [ 38 ] [ 40 ]


For a Morpholino to be effective, it must be delivered past the  cell membrane  into the cytosol of a cell. Once in the cytosol, Morpholinos freely diffuse between the cytosol and nucleus, as demonstrated by the nuclear splice-modifying activity of Morpholinos observed after microinjection into the cytosol of cells.  Different methods are used for delivery into embryos, into cultured cells or into adult animals. A  microinjection  apparatus is usually used for delivery into an embryo, with injections most commonly performed at the single-cell or few-cell stage; [ 45 ]  an alternative method for embryonic delivery is  electroporation , which can deliver oligos into tissues of later embryonic stages. [ 46 ]  Common techniques for delivery into cultured cells include the Endo-Porter peptide (which causes the Morpholino to be released from  endosomes ), [ 21 ]  the Special Delivery system (no longer commercially available, used a Morpholino-DNA  heteroduplex  and an ethoxylated  polyethylenimine  delivery reagent), [ 20 ]  electroporation, [ 47 ]  or scrape loading. [ 48 ]


Delivery into adult  tissues  is usually difficult, though there are a few systems allowing useful uptake of unmodified Morpholino oligos (including uptake into  muscle  cells with  Duchenne muscular dystrophy [ 49 ]  or the  vascular endothelial  cells stressed during balloon  angioplasty [ 50 ] ). Though they permeate through intercellular spaces in tissues effectively, unconjugated PMOs have limited distribution into the cytosol and nuclear spaces within healthy tissues following IV administration. Systemic delivery into many cells in adult organisms can be accomplished by using covalent conjugates of Morpholino oligos with  cell-penetrating peptides , and, while toxicity has been associated with moderate doses of the peptide conjugates, [ 51 ] [ 52 ]  they have been used  in vivo  for effective oligo delivery at doses below those causing observed toxicity. [ 7 ] [ 53 ]  An octa-guanidinium dendrimer attached to the end of a Morpholino can deliver the modified oligo (called a Vivo-Morpholino) from the blood to the cytosol. [ 22 ] [ 54 ]   Delivery-enabled Morpholinos, such as peptide conjugates and Vivo-Morpholinos, show promise as therapeutics for viral and genetic diseases. [ 55 ]


A  multiple cloning site  ( MCS ), also called a  polylinker , is a short segment of  DNA  which contains many (up to ~20)  restriction sites  - a standard feature of engineered  plasmids . [ 1 ]  Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. The purpose of an MCS in a plasmid is to allow a piece of DNA to be inserted into that region. [ 2 ]


An MCS is found in a variety of vectors, including  cloning vectors  to increase the number of copies of target DNA, and in  expression vectors  to create a protein product. [ 3 ]  In expression vectors, the MCS is located downstream of the  promoter . [ 2 ]


In some instances, a vector may not contain an MCS. Rather, an MCS can be added to a vector. [ 4 ]  The first step is designing complementary  oligonucleotide  sequences that contain  restriction enzyme  sites along with additional bases on the end that are complementary to the vector after digesting. Then the oligonucleotide sequences can be annealed and ligated into the digested and purified vector. The digested vector is cut with a restriction enzyme that complements the oligonucleotide insert overhangs. After ligation, transform the vector into bacteria and verify the insert by sequencing. This method can also be used to add new restriction sites to a multiple cloning site.


Multiple cloning sites are a feature that allows for the insertion of foreign DNA without disrupting the rest of the plasmid which makes it extremely useful in  biotechnology ,  bioengineering , and  molecular genetics . [ 1 ]  MCS can aid in making transgenic organisms, more commonly known as a  genetically modified organism  (GMO) using genetic engineering. To take advantage of the MCS in genetic engineering, a gene of interest has to be added to the vector during production when the MCS is cut open. [ 5 ]  After the MCS is made and ligated it will include the gene of interest and can be amplified to increase gene copy number in a bacterium-host. After the bacterium replicates, the gene of interest can be extracted out of the bacterium. In some instances, an expression vector can be used to create a protein product. After the products are isolated, they have a wide variety of uses such as the production of  insulin , the creation of  vaccines , production of  antibiotics , and creation of gene therapies.


One bacterial plasmid used in genetic engineering as a plasmid cloning vector is pUC18. Its polylinker region is composed of several restriction enzyme recognition sites, that have been engineered into a single cluster (the polylinker). It has restriction sites for various restriction enzymes, including  EcoRI ,  BamHI , and  PstI . Another vector used in genetic engineering is  pUC19 , which is similar to pUC18, but its polylinker region is reversed.  E.coli  is also commonly used as the bacterial host because of the availability, quick growth rate, and versatility. [ 6 ]  An example of a plasmid cloning vector which modifies the inserted protein is  pFUSE-Fc plasmid .


In order to genetically engineer insulin, the first step is to cut the MCS in the plasmid being used. [ 7 ]  Once the MCS is cut, the gene for human insulin can be added making the plasmid genetically modified. After that, the genetically modified plasmid is put into the bacterial host and allowed to divide. To make the large supply that is demanded, the host cells are put into a large fermentation tank that is an optimal environment for the host. The process is finished by filtering out the insulin from the host. Purification can then take place so the insulin can be packaged and distributed to individuals with diabetes.


Multiple loci VNTR analysis  ( MLVA ) is a method employed for the  genetic analysis  of particular  microorganisms , such as  pathogenic bacteria , that takes advantage of the  polymorphism  of  tandemly repeated   DNA sequences . A " VNTR " is a "variable-number tandem repeat". This method is well known in  forensic science  since it is the basis of  DNA fingerprinting  in humans.  When applied to bacteria, it contributes to  forensic microbiology  through which the source of a particular  strain  might eventually be traced back, making it a useful technique for  outbreak surveillance .


In a typical MLVA, a number of well-selected and characterised (in terms of  mutation rate  and diversity)  loci  are amplified by polymerase chain reaction ( PCR ), so that the size of each locus can be measured, usually by  electrophoresis  of the amplification products together with reference DNA fragments (a so-called DNA size marker). Different electrophoresis equipment can be used depending on the required size estimate accuracy, and the local laboratory set-up, from basic  agarose gel electrophoresis  up to the more sophisticated and high-throughput  capillary electrophoresis  devices. [ 1 ]  From this size estimate, the number of repeat units at each locus can be deduced. The resulting information is a code which can be easily compared to reference databases  once the assay has been harmonised and standardised. [ 2 ] [ 3 ]


MLVA has become a major first line typing tool in a number of pathogens where such an harmonisation could be achieved, including  Mycobacterium tuberculosis , [ 4 ]   Bacillus anthracis , [ 5 ]   Brucella . [ 6 ] [ 7 ]


NAIL-MS  (short for  nucleic acid   isotope  labeling coupled  mass spectrometry ) is a technique based on mass spectrometry used for the investigation of nucleic acids and its  modifications . It enables a variety of experiment designs to study the underlying mechanism of RNA biology  in vivo . For example, the dynamic behaviour of nucleic acids in living cells, especially of  RNA modifications , can be followed in more detail. [ 1 ]


NAIL-MS is used to study RNA modification mechanisms. Therefore, cells in culture are first fed with stable isotope labeled nutrients and the cells incorporate these into their biomolecules. After purification of the nucleic acids, most often  RNA , analysis is done by mass spectrometry. Mass spectrometry is an analytical technique that measures the  mass-to-charge ratio  of  ions . Pairs of chemically identical nucleosides of different stable-isotope composition can be differentiated in a mass spectrometer due to their mass difference. Unlabeled  nucleosides  can therefore be distinguished from their stable isotope labeled  isotopologues . For most NAIL-MS approaches it is crucial that the labeled nucleosides are more than 2  Da  heavier than the unlabeled ones. This is because 1.1% of naturally occurring carbon atoms are  13 C isotopes. In the case of nucleosides this leads to a mass increase of 1 Da in ~10% of the nucleosides. This signal would disturb the final evaluation of the measurement.


NAIL-MS can be used to investigate RNA modification dynamics by changing the labeled nutrients of the corresponding growth medium during the experiment. Furthermore, cell populations can be compared directly with each other without effects of purification bias. Furthermore, it can be used for the production of biosynthetic isotopologues of most nucleosides which are needed for quantification by mass spectrometry and even for the discovery of yet unknown RNA modifications. [ 2 ] [ 3 ] [ 4 ]


In general, cells are cultivated in unlabeled or stable (non-radioactive) isotope labeled media. For example, the medium can contain glucose labeled with six  carbon-13  atoms ( 13 C) instead of the normal  carbon-12  ( 12 C). Cells growing in this medium, will, depending on model organism, incorporate the heavy glucose into all of their RNA molecules. Thereafter, all  nucleotides  are 5 Da heavier than their unlabeled isotopologues due to a complete carbon labeling of the ribose. After cultivation and appropriate labeling of the cells, they are generally harvested using phenol/chloroform/guanidinium isothiocyanate. Other extraction methods are possible and sometimes needed (e.g. for yeast). RNA is then isolated by  Phenol-Chloroform extraction  and  iso -Propanol  precipitation. Further purification of specific RNA species (e.g. rRNA, tRNA) is usually done by  size-exclusion chromatography  (SEC) but other approaches are available as well. For most applications the final product needs to be enzymatically digested to nucleosides before analysis by  LC-MS . Therefore, digestion enzymes such as  benzonase , NP1 and  CIP  are used. [ 5 ] [ 6 ]  Typically, a  triple quadrupole  in MRM mode is used for the measurements.


How the labeling of RNA molecules is achieved depends on the model organism. For  E.coli  ( bacteria ) the minimum medium M9 can be used and supplemented with the stable isotope labeled variants of the needed salts. This enables labeling with  13 C-carbon,  15 N-nitrogen,  34 S-sulfur and  2 H-hydrogen. [ 7 ]  In  S.cerevisiae  ( yeast ) there are currently two possibilities: First, the use of commercially available complete growth medium, which enables labeling with  13 C-carbon and/or  15 N-nitrogen and second the use of minimal YNB medium which has to be supplemented with several  amino acids  and  glucose  which can be added as stable isotope labeled variants in order to achieve  13 C-carbon,  15 N-nitrogen and  2 H-hydrogen labeling of RNA. [ 8 ]


While labeling in model organisms like  E.coli  and  S.cerevisiae  is fairly simple, stable isotope labeling in  cell culture  is much more challenging as the composition of the growth media is much more complex. Neither the supplementation of stable isotope labeled glucose nor the supplementation of stable isotope labeled variants of simple precursors of nucleoside biosynthesis such as  glutamine  and/or  aspartate  is sufficient for a defined mass increase higher than 2 Da. Instead, most cells kept in cell culture can be fed with stable isotope labeled methionine for labeling of methyl groups and with stable isotope labeled variants of adenin and uridine for labeling of the nucleoside's base body. [ 9 ]  Special care must be taken when supplementing the medium with FBS (fetal bovine serum), as it also contains small metabolites used for the biosynthesis of nucleosides. The use of dialyzed FBS is therefore advisable when defined labeling of all nucleosides is desired.


With NAIL-MS different experiment designs are possible.


NAIL-MS can be used to produce stable isotope labeled internal standards (ISTD). Therefore, cells are grown in medium which results in complete labeling of all nucleosides. The purified mix of nucleosides can then be used as ISTD which is needed for accurate absolute quantification of nucleosides by mass spectrometry. This mixture of labeled nucleosides is also referred to as SILIS (stable isotope labeled internal standard). [ 10 ]  The advantage of this approach is, that all modifications present in an organism can thereby be biosynthesized as labeled compounds. The production of SILIS was already done before the term NAIL-MS emerged.