For each strand, we provide a primer , which is a short piece of DNA that sticks to one end of the strand. An enzyme is added. This is a specific type of protein called a "DNA polymerase" that can "read" the bases on one strand and can attach the complementary base to the growing strand. The polymerase "walks" down the template strand and creates its exact complement as it goes. Note that the refractive index change is not due to the film thickness variation, since all the films were fabricated at a nearly same thickness, and therefore it is due to the DNA denaturation.
The refractive index decrease in denaturated DNA thin solid film is quite predictable, since DNA denaturation breaks the nucleobase bonds, and subsequently, the net density decreases [ 35 ] in the solid thin films.
The refractive index of a solid medium is directly proportional to its density [ 36 ], and therefore, DNA denaturation could result in a density decrease and subsequently a refractive index decrease. We plotted the refractive index change as a function of NaOH concentration in Fig. The refractive index difference between the pristine DNA film and the film with 7.
When the NaOH concentration further increased beyond 7. We did measure the refractive index of DNA thin film with a higher NaOH concentration and confirmed that it continued to decrease but the film thickness was much thinner.
However, NaOH embedded in the DNA solid thin films could contribute to the refractive index changes due to its inherent refractive index [ 37 , 38 ]. Variation of the refractive index with the external temperature, the thermo-optic effect, is a very fundamental mechanism to add novel functionalities in photonic devices providing spectral tuning, switching, and sensing applications.
The results are summarized in Fig. We also observed a monotonic decrease of the film thickness as the temperature increased, and after the first cycle, the film thickness slightly decreased due to additional drying of the DNA film, which is also consistent with prior reports [ 10 , 11 ].
We repeated measurements in successive temperature cycles and observed no further significant changes in the thermo-optic coefficients and the film thickness within experimental errors after the second cycle. The addition of NaOH and the subsequent increase in the level of DNA denaturation especially showed a good linear correlation with the magnitude of the thermo-optic coefficient, which could be attributed to both the random coil structure of ss-DNA and NaOH ingredients in the DNA thin films.
Thermally induced changes in the refractive index and the film thickness of DNA thin solid film with various NaOH concentrations in precursor solutions.
We have successfully developed a new method to precisely control the refractive index and thermo-optic coefficient of DNA thin solid film by adopting a denaturation process in the aqueous solution precursor to unwind double-stranded DNAs ds-DNAs into single-stranded DNAs ss-DNAs.
These ss-DNAs were irreversibly immobilized in thin solid films by an optimal spin-coating process to systematically alter the physical properties of the film.
We experimentally confirmed a high potential to make all-DNA photonic devices by using unique refractive index control of DNA film by the denaturation process. Watson and F. Singh, N. Sariciftci, J. Grote, and F. Hagen, W. Li, A. Steckl, and J. Steckl, H. Spaeth, H. You, E. Gomez, and J. News 22 , 34—39 Kawabe, L.
Wang, S. Horinouchi, and N. Heckman, R. Aga, A. Rossbach, B. Telek, C. Bartsch, and J. Khazaeinezhad, S. Kassani, B. Paulson, H. Jeong, J. Gwak, F. Rotermund, D. Yeom, and K. Kulkarni, B. Kim, S. Dugasani, P. Joshirao, J. Kim, C. Vyas, V. Manchanda, T. Kim, and S. Hong, W. Jung, T. Nazari, S. Song, T. Quan, and K. Jung, H. 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. Methods A series of physical and chemical denaturation methods were implemented on well-defined bp dsDNA fragment. Results Heating, beads mill, and sonication bath did not show any denaturation for 30 minutes. Conclusions Among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments.
Heating Two variable methods i. Beads Mill Two sizes i. Indirect Sonication Two ways of indirect sonication i.
Direct Sonication Direct probe sonication with probe horn was applied to facilitate the denaturation of the DNA. Alkaline Various concentrations of NaOH 0. Formamide The formamide in liquid form of molecular biology grade Renaturation of the DNA Denatured by Chemical Treatments The denatured DNA can reformulate hydrogen bonds between complementary single strand, making it likely to reform double helix structure again. Evaluation of Renaturation The renaturation efficiency was also calculated based on Equation 2.
Open in a separate window. Figure 1. Figure 2. Figure 3. Beads Mill 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 ]. Indirect Sonication The ultrasonic bath has been used for lysing cells and breaking the chromosome DNA for lower stream experiments such as PCR or microarray [ 22 ].
Sonication The direct probe sonication was carried out to denature the DNA. Formamide 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.
References 1. A novel automated assay with dual-color hybridization for single-nucleotide polymorphisms genotyping on gold magnetic nanoparticle array. Anal Biochem. The heat of denaturation of DNA. J Am Chem Soc. Ando T. A nuclease specific for heat-denatured DNA in isolated from a product of Aspergillus oryzae.
Biochim Biophys Acta. Effect of diethylsulfoxide on the thermal denaturation of DNA. Though DNA melting is a fairly simple and straightforward process, it is generally not used when accuracy is required. Heated DNA denaturation is considered to be less accurate than DNA sequencing and is used for more broad scope applications. This type of denaturation may also be used within the polymerase chain reaction.
Apart from heating, chemical denaturation can also be achieved through the use of NaOH. As the concentration of NaOH used is reduced, the process will take longer -- but the DNA can still be fully denatured.
NaOH has been shown to be one of the most effective and reliable methods of complete denaturing. Other chemicals, such as formamide, are not able to denature DNA as rapidly or as reliably. Because NaOH can be used at a variety of concentrations, it is also able to be scaled quite easily.
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