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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. Geiduschek [ 16 ] also observed the rapid DNA renaturation when the temperature decreased below the T m. This problem can be fixed by the more sophisticated setup of heating system along with the elucidation of kinetics for this potentially rate-dependent reaction.
On the other hand, a number of researchers used the heating and cooling cycle technique to manipulate the DNA in PCR [ 6 , 11 , 17 , 18 ]. This open system allows for the temperature of the denatured DNA to drop when it is transferred to the hybridization buffer. The beads mill method is one of the well-known physical disruption techniques used to break down the microbial cell wall for the DNA extraction [ 19 - 21 ].
In this study, we prolonged the time of the beads mill to denature the DNA fragment and monitored the denaturation capability over time. The strong denaturation capability of the beads mill method may allow us to combine the cellular lysis and DNA denaturation; thus simplifying the sample preparation procedure for the hybridization using whole cells. However, as shown in Figure 1B , beads mill methods with 0. This incomplete denaturation of the DNA indicated that the disruption technique using the glass beads was not strong enough to break the double helix structure of the DNA.
The ultrasonic bath has been used for lysing cells and breaking the chromosome DNA for lower stream experiments such as PCR or microarray [ 22 ]. In order to simplify DNA hybridization from whole cells, the denaturation capability of the ultrasonic bath was investigated. Probe sonication is a more fierce approach which is also popular in cell or DNA disruption. For the indirect probe sonication in this study, the absence of direct contact of the probe and DNA made the DNA free of possible contamination.
However, the disruptive capability of the indirect probe sonication decreased significantly. Hence, the indirect sonication methods are not suitable for DNA denaturation. The direct probe sonication was carried out to denature the DNA. 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.
This is likely due to the combinational effects based on heating as well as the movements of the shock waves generated during the sonication. Sonication is a commonly used fragmentation method [ 7 , 23 ], but it has not yet been used for denaturing DNA. Therefore, we believe that the increase of the temperature was a crucial part during sonication treatment for DNA denaturation. Moreover, unlike the heating method, sonication may also disturb the denatured DNA and prevent the rapid renaturation.
Therefore, among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments. At an alkaline pH, OH- groups are predominant. They remove the hydrogen- bonds-contributing protons from guanine and thymine, thus breaking the hydrogen bonds between the two oligonucleotides [ 27 ]. The result shown in Figure 2A is consistent with the previous studies, where the NaOH concentration of 0. Those concentrations are within the range between our second highest 0.
The formamide is known for its ability to lower the T m of DNA [ 30 ], thus the DNA denatures in the lower temperature than the melting temperature. McConaughy et al. According to those studies, the original T m This means the formamide was not able to decrease the T m to ambient temperature i.
As shown in Figure 2C , the denaturation capability of the DMSO became more pronounced with higher concentrations at ambient temperature. According to Markarian et al.
Using the same interpretation, the T m The result shown in Figure 3 indicates the pH change is critical to the success of denaturation when the alkaline method is being used. The renaturation of the DNA is likely due to the decrease in the chemical concentration under hybridization conditions as compared to the same concentration under denaturation conditions.
In summary, a series of physical and chemical denaturation methods were implemented on bp dsDNA fragment. National Center for Biotechnology Information , U. Journal List Environ Health Toxicol v. Environ Health Toxicol. Published online Sep Author information Article notes Copyright and License information Disclaimer. Received Jun 12; Accepted Aug 8.
This article has been cited by other articles in PMC. Generally, they are important in the number of bioassays, which involve DNA hybridization, such as membrane hybridization, microarrays , PCR , etc. It occurs as a result of breaking hydrogen bonds. Besides, the denaturation of the double helix can occur in two methods; the physical denaturation and chemical denaturation. Physical denaturation is mainly done by heating and sonication.
Technically, this is done by providing enough kinetic energy to breakdown hydrogen bonds between the base pairs. Subsequently, this results in the disruption of Watson and Crick base pairs and the separation of the double-stranded helix into two single strands. In this process, the characteristic temperature that melts the DNA double helix is known as the melting temperature Tm.
Also, Tm increases with the increasing G-C content as guanine and cytosine form three hydrogen bonds during base pairing, while cytosine and thymine form only two hydrogen bonds. Generally, the degree of denaturation can be spectrophotometrically determined by monitoring the absorbance of light at nm. In biology, examples of biomolecules that denature are proteins and nucleic acids e.
Renaturation in molecular biology refers to the reconstruction of a protein or nucleic acid such as DNA to their original form especially after denaturation. This process is therefore the inverse of denaturation.
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