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   Phenol as a cleaning agent for DNABased on the discovery that phenol is suitable to extract proteins from aqueous solutions (Grassmann & Deffner, 1953), Kirby reported phenol as an agent for nucleic acid purification (Kirby, 1956). Using a phenol-water mixture he extracted a tissue homogenate and showed that RNA was separated in the aqueous phase, while proteins associated with DNA were transferred to the interphase. Shortly after, Kirby found, that in the presence of certain anionic salts (e.g. p-aminosalicylate and sodium benzoate) both nucleic acid species, DNA and RNA, are enriched in the aqueous phase (Kirby, 1957). Today, Kirby's method is still up-to-date, apart from a few adaptions (anionic detergents like SDS replaced the anionic salts, addition of chloroform and isoamyl alcohol, and equilibration of the pH). Polar, hydrophilic compounds like DNA, RNA and proteins commonly dissolve best in polar solvents (with water as the solvent offering maximum polarity). But in contrast to nucleic acids, proteins provide a number of non-polar structures as well. The non-polar side chains of phenylalanine, leucine, isoleucine, valine, proline, methionine and alanine enable the protein to stay in solution when exposed to a less polar or even non-polar solvent. The proteins rearrange exposing the non-polar side chains to the surface, while the charged and polar residues become buried inside the protein complex. These features enable the extraction of proteins out of an aqueous phase by a less or even non-polar solvent. Phenol is clearly less polar than water despite its electronegative oxygen atom; because the phenyl ring renders the electron density spread all over the molecule but not concentrated on the oxygen atom.For DNA isolation, the phenol has to be pH-equilibrated with tris to a final pH of >7.8 to ensure that the DNA is negatively charged and therefore insoluble in the organic phase. Starting with the cell lysate, an equal volume of tris-buffered phenol-chloroform (1:1), or tris-buffered phenol-chloroform-isoamyl alcohol (25:24:1) is added and the solution is mixed by vortexing (small DNA molecules of <10 kb), gently shaking (10-30 kb) or slowly inverting or rotating (>30 kb). Chloroform efficiently denatures proteins, avoids the retention of water in the organic phase and improves the phase separation by increasing the density of the organic phase. The addition of a small volume of isoamyl alcohol reduces foaming during the extraction process, and, aiming at RNA isolation, guarantees the deactivation of RNases (Green & Sambrook, 2012).Centrifugation accelerates the separation of the two phases, resulting in the aqueous phase with the lower specific gravity on top. But caution: A high salt content or high amounts of sucrose in the aqueous solution can result in an inversion of the two phases! So it is highly recommended to make sure the right solution is further processed. Since equilibrated phenol commonly contains 8-hydroxyquinoline as a stabilizer, the organic phase can be identified by its yellow color. After organic extraction, DNA (and RNA, if no RNase A has been added during lysis) is still in the aqueous phase while denatured proteins have moved into the interphase and lipids have been transferred to the organic phase.After repeated extractions with the phenol-containing solvent, the DNA-containing aqueous phase is extracted a few times with chloroform (or chloroform/isoamyl alcohol), to remove residual phenol. Finally, the pure DNA is precipitated from solution using ethanol or isopropanol.AppliChem   s products for Phenol-Chloroform Extraction of DNAProd. No.Description Comment A1153Phenol equilibrated, stabilizedPure phenol is a colorless and crystalline substance. Liquefied phenol is suscep  tible to oxidation (especially when the pH is equilibrated with Tris) and the phenolic oxidation products introduce strand breaks into nucleic acid molecules and promote cross-linking. Phenol solutions should be clear and colorless, pink or brownish solutions should be discarded. To prevent oxidation, 8-hydroxyquinoline is frequently added to liquefied phenol. The shelf life of equilibrated and 8-hydroxyquinoline-stabilized phenol is approximately 9 months. As a positive side effect, 8-hydroxyquinoline partially inhibits ribonucleases.A0971Phenol equilibrated, non stabilizedA1624Phenol water-saturated, stabilizedA1578Phenol water-saturated, non-stabilizedA1594Phenol crystalline A0889Phenol equilibrated, stabilized : Chloroform : Isoamyl alcohol 25:24:1 A3691ChloroformA2610Isoamyl alcoholA21071-Bromo-3-chloropropane Bromochloropropane is less toxic than chloroform and forms a tighter interphase (Chomczynski & Mackey, 1995a). back The all in one solution: Acidic guanidinium thiocyanate-phenol extractionAdmittedly, this technique does not really aim at DNA isolation in the first place, but nevertheless it is possible to use it on this purpose. And since this technique offers interesting possibilities it should be mentioned in this article.Invented by Chomczynski & Sacchi in 1987 for isolation of RNA, acidic guanidinium thiocyanate-phenol extraction allows cell lysis and successive separation of RNA, DNA and proteins using one reagent. The technique combines the effect of chaotropic salts on structure and solubility of macromolecules with the extraction properties of acidic phenol. Commercialized under names like Trizol®, TRI REAGENT® and TRItidy™ G, Chomczynski's reagent conquered the laboratories and still belongs to the number one choices for RNA isolation. The birth of monophasic reagentsThe history of acidic guanidinium thiocyanate - phenol extraction (or the ªsingle-stepº method) invented by Chomczynski & Sacchi is in first place the history of RNA isolation. In 1951, the usage of guanidinium chloride in RNA isolation was firstly described (Volkin & Carter, 1951). After Kirby's introduction of phenol as a deproteinization reagent, guanidinium salts temporarily seem to lose their importance, but ªguanidinium chloride in the isolation of nucleic acidsº was focused again in the 1960s (Cox 1968). In 1979, Chirgwin et al. published a method to isolate undegraded RNA from ribonuclease-rich tissues employing guanidinium thiocyanate instead of guanidinium chloride. In 1987, Chomczynski & Sacchi initially used a combination of guanidinium thiocyanate and phenol-chloroform extraction for RNA isolation. Based on the same principle, the method was expanded for simultaneous isolation of RNA, DNA and proteins (Chomczynski, 1993). Two years later, further modifications were published employing bromochloropropane (Chomczynski & Mac  key, 1995a) which is less toxic than chloroform and forms a tighter interphase, and isopropanol (Chomczynski & Mackey, 1995b) for improved isolation of RNA from polysaccharide- and proteoglycan-rich sources. The development finally also focused on the isolation of genomic DNA (Chomczynski et al., 1997).The monophasic solution contains water, phenol, guanidinium thiocyanate (also often referred to as guanidine thiocyanate), b-mercaptoethanol and a detergent. The chaotropic salt guanidinium thiocyanate lyses the cells, denatures the released macromolecules and inactivates RNases and other enzymes. As a detergent, lauroylsarcosine (ªsarkosylº) is a good choice, since, in contrast to SDS, lauroylsarcosine shows high solu   ility in chaotropic high salt   uffers. Addition of sarkosyl to cell or tissue homogenates improves the purity of the RNA isolated   y guanidinium salts and reduces foaming during homogenisation (MacDonald et al., 1987).After lysis of the cell in the reagent (e.g.   y repetitive pipetting), chloroform (or alternatively   romochloropropane) is added, leading to the generation of a second phase. While DNA and proteins enrich in the newly formed organic phase and the interphase, RNA is selectively retained in the aqueous phase. RNA, DNA and proteins are isolated   y alcohol precipitation.Why is hydrophilic DNA transferred into the organic phase? And how is it possi   le that RNA, in contrast, is not extracted   y the phenol-chloroform mixture? DNA and RNA seem to   e very similar at first sight and, under neutral conditions (pH 7-8)   oth molecules remain in the aqueous phase as expected. However, usage of non-equili   rated, and therefore acidic phenol solution, ena   le the enrichment of DNA molecules in the non-polar organic phase. So why do these two types of nucleic acids   ehave differently under acidic conditions? The answer mainly lies in the structural differences   etween DNA and RNA: at the prevailing pH of 4-5, the phosphate groups of the dou   led stranded (and less acidic) DNA are protonated and the affinity of the now non-charged (   ut still dou   le stranded) DNA molecules to the organic solvent strongly increases. The phosphate   ack   one of the single stranded RNA is largely protonated as well,   ut due to the exposure of the purine and pyrimidine   ases, the RNA is a   le to form hydrogen   onds with the surrounding water molecules (Zum   o, 2011). As a result, RNA does not lose its hydrophilic properties and still prefers the aqueous phase.AppliChem's monophasic reagents: acidic guanidinium thiocyanate-phenol extractionProd. No.Description Comment A2867RNAtidy GReady-to-use solution for the isolation of small and large RNA species (0.1 - 15 k   ) from   iological material; provides high purity RNA (DNA and protein-free); suited for small and large samples.A4051TRItidy G™Ready-to-use solution for sequential isolation of RNA, DNA and proteins.A3418 DNA - Isolation reagent for genomic DNA Phenol-free ready-to-use reagent for the isolation of genomic DNA from human, animal (incl. mouse tail), plant, yeast,   acterial and viral srcin.Fast desalting: Ethanol precipitationToday, ethanol precipitation is often used as the final step in DNA isolation   y organic extraction or anion-exchange chromatography. The possi   ility to precipitate DNA out of solution ena   les not only removal of alcohol solu   le salts, residual organic solvents and detergents; it also offers an opportunity to concentra  te DNA.In neutral aqueous solutions, the negatively charged phosphate   ack   one of DNA molecules is saturated with the highly polar water molecules that prevent interaction with cationic molecules. Increasing concentrations of ethanol lead to a disruption of the hydrate shell and allow the formation of ionic   onds   etween the phosphate groups and positively charged ions. As a consequence, in solutions containing at least 65% ethanol and a sufficient amount of cations, the previously negatively charged DNA molecules are neutralized. The loss of charge minimizes the repulsive forces   etween the molecules and finally makes DNA to precipitate. The accurate salt concentration is crucial to ensure that on the one hand, all DNA is recovered from solution, and on the other hand, the anionic salts do not co-precipitate. To improve the efficiency, the target nucleic acids often are co-precipitated with nuclease-free inert ªcarriersº such as glycogen or linear polyacrylamide.The most common salts used in ethanol precipitations are ammonium acetate, sodium acetate and sodium chloride.A sodium acetate solution at acidic pH of 5.2 is the standard reagent for nucleic acid precipitation. Sodium chloride is the first choice if SDS is included in the sample ensuring its solu   ility in the presence of alcohol and ena   ling the precipitation of detergent-free DNA. If the sample is contaminated   y dNTPs or oligosaccharides (which includes DNA samples o   tained from agarose gels   y agarase digestion), ammonium acetate is most suita   le since the ammonium cations prevent co-precipitation of these two species (source: Green & Sam   rook, 2012).Isopropanol can   e used alternatively for DNA precipitation; it is mostly used for large volumes since only half of the quantity is required to dehydrate the DNA molecules. Unfortunately, the solu   ility of salts in solutions with 35% isopropanol is reduced compared to 65% ethanol, increasing the risk of salt co-precipitations. Furthermore, it is less volatile than ethanol and therefore harder to remove, increasing the risk of alcohol carry-over into the final sample.AppliChem's products for alcohol precipitation of nucleic acidsProd. No.DescriptionA3678Ethanol a   soluteA39282-PropanolA4555Sodium acetate anhydrousA2936Ammonium acetateA6587DNA-PrecipitAid.Synthetic produced polyacrylamide carrier for precipitating picogram amounts of nucleic acids. DNA-PrecipitAid ena   les to completely recover DNA fragments larger than 20   ase pairs. DNA-PrecipitAid can also   e used to precipitate RNA with ethanol. Coprecipitant of choice for PCR/RT-PCR, since no contamination   y nucleic acids is detecta   le.   ack The convenient method: Commercial kits employing pure silica and anion exchange

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Jul 23, 2017
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