Salt is often used in addition to an acid for the full denaturation of DNA, and it may also be used in conjunction with heat. Salt is not usually used as the sole process of denaturation -- it's usually used alongside other chemicals such as isopropanol and ethanol.
This process is able to be used on larger volumes of DNA, which makes it less useful for highly accurate and specific work, but more useful for scaling up and processing DNA in larger quantities. Though there are many techniques associated with DNA denaturation, the end result is the same: the bonds between the strands are broken and new molecules are formed, which can then be compared as desired.
The ideal process of DNA denaturation depends on what the DNA needs to be used for, how accurate and specific the comparisons need to be, and the volume of material that has to be processed. In general, both heat and salt denaturation can be easily scaled and used with larger quantities, while NaOH denaturation may be slightly more accurate and useful in smaller quantities.
This assay is suitable for the simple and rapid estimation of protein concentration. This assay is based on a single Coomassie dye based reagent. The binding of protein to the dye results in a change of color from brown to blue. The change in color density is proportional to protein concentration. Protein estimation can be performed using as little as 0. Denaturation of DNA double helix takes place by the following denaturating agents:. This denaturation is very abrupt and is accelerated by chemical reagents like urea and formamide.
The chemicals enhance the aqueous solubility of the purine and pyrimidine groups. This separation of double helix is called melting as it occurs abruptly at a certain characteristic temperature called denaturation temperature or melting temperature T m. The abruptness of the transition indicates that the DNA double helix is highly cooperative structure, held together by many reinforcing bonds.
T m is analogous to the melting point of crystal. The T m value depends on the nature of the DNA. This relationship between T m and G—C content arises due to guanine and cytosine form three hydrogen bonds when base paired, whereas adenine and thymine form only two.
If denaturation is followed spectrophotometrically by monitoring the absorbance of light at nm, it is observed that the absorbance at nm increases as the DNA become denatured, a phenomenon known as the hyperchromatic effect or hyperchromacity or hyperchromism.
This is due to un-stacking of base pairs. Double-stranded DNA shows a strong positive rotation which highly decreases with denaturation. This change is analogous to the change in rotation observed when the proteins are denatured. The solutions of native DNA exhibit high viscosity because of the relatively rigid double helical, long and rod like character of DNA molecule. Denaturation causes a marked decrease in viscosity. If melted DNA is cooled it is possible to reassociate the separated strands, a process known as renaturation.
However, a stable double-stranded molecule may be formed only if the complementary strands collide in such a way that their bases are paired precisely. But renaturation may not be precise if the DNA is very long and complex. Thus the rate of renaturation renaturation kinetics can give information about the complexity of a DNA molecule.
Complete denaturation is not a readily reversible process. Rapid cooling does not reverse denaturation, but if the cooled solution is again heated and then cooled slowly, renaturation takes place. Denaturation of DNA double helix can also be brought about by certain chemical agents such as urea and formamide.
These chemical reagents enhance the aqueous solubility of the purine and pyrimidine groups. The T m value is lowered by the addition of urea. Denaturation also occurs at acidic and alkaline solutions in which ionic changes of the purine and pyrimidine bases can occur.
A series of physical and chemical denaturation methods were implemented on well-defined bp dsDNA fragment. The degree of each denaturation was measured and the most suitable denaturation method was determined. DNA renaturation tendency was also investigated for the suggested denaturation method.
Heating, beads mill, and sonication bath did not show any denaturation for 30 minutes. However probe sonication fully denatured DNA in 5 minutes. Among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments.
It serves a basis for a number of bioassays such as microarray, membrane hybridization, and fluorescence in situ hybridization. Even though the denaturation is a key reaction that determines the success of DNA hybridization based bioassays, no systematic characterization of denaturation method for dsDNA has been attempted thus far.
Substantial studies have described the methods of DNA denaturation, including heating [ 1 - 3 ], dimethyl sulfoxide DMSO [ 4 , 5 ], and sonication [ 6 , 7 ]. In the above methods, the heating at high temperature e. In this study, a series of physical denaturation heating, heating with cold shock, beads mill with 0.
Well-defined dsDNA of 86 bp was used as a template of each denaturation. The result presented in this study will provide useful data to any DNA hybridization based applications. The DNA used for the denaturation and renaturation was part of the eaeA gene, which has been used as a common biomarker for detection of a notorious pathogen, E.
The freeze dried E. A series of simple physical methods i. Detailed descriptions of the four physical methods are described below. Two variable methods i. The Final A of each group of sample were immediately recorded after they were removed from the incubator.
Two sizes i. The triplicate DNA samples were disrupted with the beads for 1, 5, 10, 20, and 30 minutes at 2, rpm with a Disruptor Genie bead-beater Scientific Industries ; followed by centrifuging at 3, rpm for 30 seconds to precipitate out the beads. Two ways of indirect sonication i. For the indirect probe sonication method, the centrifuge tube was attached on the wall of a 5 mL well, which is filled with deionized water. The DNA was subject to the indirect sonication at 10 W, For the ultrasonic bath, the bottom of the centrifuge tubes were immerged into the water of the ultrasonic bath Branson Ultrasonic Cleaner; Branson Ultrasonics, Danbury, CT, USA at 40 kHz for the same amount of the time.
Ice was added to maintain the deionized water at room temperature during the indirect sonication treatments. Direct probe sonication with probe horn was applied to facilitate the denaturation of the DNA. The temperature was monitored with a glass laboratory thermometer after each time interval to monitor the temperature change during the sonication.
The chemical denaturation methods looked into in this study includes a variety of concentrations of NaOH, formamide, and DMSO at ambient temperature. Detailed descriptions for the three methods used are described in the following sections. Various concentrations of NaOH 0. The NaOH was serially diluted from 1 to 0.
The mixture was homogenized with continuous pipetting and incubated at ambient temperature for 1, 2, 5, 10, 20, and 30 minutes, after which the Final A was measured using the NanoDrop spectrophotometer.
The formamide in liquid form of molecular biology grade The mixtures were homogenized gently with pipette and incubated at room temperature, and the Final A s were recorded with the same manner at 1, 2, 5, 10, 20, and 30 minutes. Solutions were again homogenized gently, incubated at room temperature, and applied for the absorbance measurement at 1, 2, 5, 10, 20, and 30 minutes. An simplified equation developed previously by Wang and Son [ 14 ], in which denaturation percentages were calculated with the numerical change of the absorbance of the DNA at nm.
Where, Initial and Final A are the absorbance of the DNA at nm before and after any denaturation treatments, respectively. Blank A is the absorbance of the free water or reagents after physical or chemical treatments.
The Final A contained the absorbance caused by the physical methods i. The denatured DNA can reformulate hydrogen bonds between complementary single strand, making it likely to reform double helix structure again. This process is called as renaturation. The instant concentration change of the reagents when the denatured DNA is added to the hybridization buffer may cause the unwanted renaturation.
Therefore the tendency of renaturation was investigated during the hybridization process after the chemical treatments. The phosphate buffer 0. It was confirmed that the phosphate buffer had no absorbency at nm and would therefore not interfere with the absorbance of the DNA at nm.
The renaturation efficiency was also calculated based on Equation 2. A series of physical methods were applied to the DNA fragment in order to investigate the denaturation efficiency of each method. As shown in Figure 1 , the denaturation efficiency, presented as a percentage, was plotted against the time of the DNA being treated with physical methods. Beads mill with both 0. There was no denatured DNA over 30 minutes of ultrasonic bath treatment Figure 1C open circle and indirect sonication Figure 1C closed circle.
However, as shown in Figure 1D , the denaturation capability of the sonication increased with the time, and achieved complete denaturation capability after being sonicated for seconds. DNA denaturation by the physical treatments. The treatments include A heating only closed circles and heating with cold shock open circles , B beads mill with 0.
Chemical methods were also applied to denature the DNA and their denaturation efficacies are presented in Figure 2. The denaturation by 0. The 0. DMSO also showed the efficient denaturation result Figure 3. DNA denaturation by the chemical treatments. DMSO, dimethyl sulfoxide. The possibility of renaturation of already denatured DNA was monitored over time under the hybridization conditions. The sudden drop of temperature can cause an immediate renaturation effect of the already denatured DNA, making the absorption at nm returned to be the same as the one before heating.
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