Contents
- Denaturation of DNA
- Renaturation of DNA
- DNA Denaturation through Heat | Thermal Denaturation of DNA
- DNA Denaturation through NaOH Treatment
- DNA Denaturation through Salt
- Which PCR step causes the denaturation of double stranded DNA
- Effect of urea on DNA denaturation
- Conclusions
- Interview Q & A
What is Denaturation of DNA
During the replication of DNA and other different phenomenon in which active participation of DNA is observed with the separation of strands of DNA double helix.
Strands of the DNA double helix can be segregated simply by heating dissolved DNA at high temperatures. The stands of DNA separate because of the disruption of the hydrogen bonds which holds together the complementary nitrogenous bases present on the opposite strands. This phenomenon is known as DNA melting.
The extent of melting is determined by the melting temperature (Tm) which is characterized as the temperature at which almost 50% of the double helical structure is lost.
Strands of DNA double helix can also be separated by the addition of alkali or acid, hydrogen bonds are disrupted by ionization.
Double helical DNA absorbs less UV light as compared to the single strand of DNA because of the base pair stacking. When DNA melts, percentage of single stranded DNA increases which results into a increase in the UV absorption profile. This phenomenon is known as hyperchromicity. Thus, the extent of denaturation or melting can be monitored by measuring absorbance at 260 nm.
Renaturation of DNA
Segregated strands of DNA start to anneal immediately when temperature drops below the Tm value. This annealing cycle is often called as renaturation. The property of denaturation and renaturation is very important for the optimum biological functioning of the DNA.
In the cellular environment, the strands of DNA are segregated by the action of enzymes like helicase and topoisomerase on the expense of energy (ATP) not by the action of heat or extreme pH.
The capacity of DNA to reversibly denature and reanneal gives evidences for the gene expression and about the position and structure of a gene.
Say for example, if the DNA particles from two distinct living organisms denatured and permitted to reanneal or hybridize with one another. The strands will form DNA hybrids if the sequences are similar to each other. The extent of hybridization indicates the extent of similarity between the DNA of two organisms.
Similar observations can be made with DNA and RNA inside the cell to locate genes.
Thermal Denaturation of DNA
DNA can be denatured through heat and this process is same as melting. The sample is heated until the DNA unwinding and the separation of two strands. When the strands have been separated, the DNA will then, at that point be allowed to reach a steady temperature. This process permits strands to be shaped in the DNA helix, which then, at that point creates complementary pairings that can be taken as markers.
Thermal denaturation is every now and again utilized when searching for the difference between distinct species. However DNA denaturation or melting is a genuinely simple and direct process, it is not utilized when precision is required. Thermal DNA denaturation is viewed as less precise than DNA sequencing and is utilized for much broader applications. This kind of denaturation may likewise be utilized inside the polymerase chain response.
DNA Denaturation through NaOH Treatment
Besides heating the DNA sample, Chemicals like NaOH can also be used to achieve denaturation of DNA. A specific concentration of NaOH can be utilized to denature DNA. As the amount of NaOH utilized is lowered, the denaturation will take longer time than expected – yet the DNA can in any case be completely denatured. NaOH has been demonstrated to be perhaps the best and efficient strategies for complete denaturation of DNA.
Different chemical agents, for example, formamide, can’t denature DNA as quickly. Since NaOH can be utilized at various concentrations, it is also easy to monitor without any problem. Moreover, the DNA that is denatured with NaOH can be renatured using a buffer solution of phosphates. DNA that is denatured through different chemical agents, like DMSO (Dimethylsulfoxide), can’t be completely renatured in this way – and this can result in the use of NaOH for more applications.
DNA Denaturation through Salt
A high amount of salt in the medium will make DNA normally denature, given the right ratio of salt. DNA denaturation using salt is like denaturation using organic solvents. generally, DNA denaturation using salt can’t be renatured. Salt is frequently utilized in place of an acid to fully denature DNA, and it might likewise be utilized along with heat. Salt isn’t generally utilized as the only agent for denaturation – it’s typically utilized with some different chemical compounds like isopropanol and ethanol.
This process can be utilized for denaturing bigger volumes of DNA, which makes it less helpful for explicit work, however more valuable for increasing and processing DNA in bigger amounts.
However there are numerous procedures related with DNA denaturation, the final product is something similar: the connections between the strands are broken and single stranded DNA is produced, which can be determined by several methods. The best method of DNA denaturation relies upon what the DNA should be utilized for, how exact and explicit the correlations should be, and the volume of material that must be processed.
As a rule, both thermal and salt denaturation can be scaled easily and utilized with bigger amounts, while NaOH denaturation might be marginally more precise and valuable for small amounts of DNA.
Which PCR step causes the denaturation of double stranded DNA
Separation of DNA strands take place in denaturation step of polymerase chain reaction (PCR) which is described in the following steps:
Effect of urea on DNA denaturation
Like Thermal and pH induced denaturation of DNA, Urea also possess the capability to denature DNA. Urea in high amounts addresses a problematic condition for nucleic acid structures. Since urea is hydrogen-bond donor as well as acceptor, it can undoubtedly denature nucleic acids. Indeed, 6–8 M urea is the critical component for denaturing polyacrylamide gel electrophoresis (dPAGE) broadly used for the separation of DNA oligonucleotides bon the basis of size. In this strategy, DNA double helices are totally denatured and travel in polyacrylamide/agrose gel as linear polymers.
To our incredible amazement, we went over a DNA particle that can shape a folded structure in 7 M urea (7MU), which stays stable during the cycle of 7MU-dPAGE. This is because of the fact that this DNA was having a very high concentration of guanine nucleotide in it and formed quadruplex structures.
Conclusions
In this article we have discussed about DNA denaturation which is very frequently exploited in the field of molecular biology as an effective tool to change, manipulate and to form recombinant DNA. We have discussed denaturation in the light of different denaturants that are known to disturb the stabilizing forces of DNA structure.
Interview Q & A
Q1 Why is DNA denatured?
Answer: As we know the stability of DNA is important for every living cell to function properly. But, there are certain occasions in which denaturation of DNA in beneficial for the cell to carry out normal life processes. DNA denatures to produce two polynucleotide strands, it is carried out by the help of specific enzymes like gyrase, topoisomerase and helicase and the denaturation is often localized.
Important cellular processes like replication and transcription of DNA requires the separation of DNA. while the denaturation phenomenon is often used is advanced molecular biology techniques like polymerase chain reaction, DNA fingerprinting etc.
Q2 What causes denaturation?
Answer: DNA strands are held together by non covalent interactions like hydrogen bonding. Breaking of these hydrogen bonds causes separation of the strands of DNA which is known as denaturation of DNA. The hydrogen bonds present between the nucleotides of DNA can be broken by several ways.
- Denaturation of DNA by Heat: Thermal denaturation of DNA
- Denaturation of DNA by extreme pH: pH induced denaturation of DNA
- Denaturation of DNA by salts: Saline induces denaturation of DNA
Q3 Factors affecting denaturation of DNA
Answer: The denaturation of DNA depends upon the following factors
- Temperature: DNA of every organism has a fixed melting/denaturation temperature
- pH: DNA denatures at extreme pH
- Osmolarity and Salinity: Extreme salt concentration also induce denaturation of DNA
- Guanine-Cytosine content: Higher the GC content in the DNA, higher will be the melting temperature
- Adenine-Thymine content: Higher the AT in the DNA, lower will be the melting temperature
Q4 Denaturation temperature of DNA
Answer: When the DNA solution is heated above 90oC, the increase in kinetic energy is enough to disturb the non covalent hydrogen bonds present between the two strands of DNA. These non-covalent interactions are responsible for stabilizing DNA. Disturbance of these forces leads to the denaturation of DNA. Generally the melting temperature of DNA of every organism is above 90oC.
Q5 At what temperature do denaturation of DNA double helix takes place?
Answer: Generally, the melting or denaturation temperature of DNA of an organism is near or above 90oC.
Q6 How does the denaturation of DNA help to analyse its structures?
Answer: When the denaturation of DNA starts, the UV absorption at 260 nm increases gradually and reaches to maximum till the two strands of DNA are fully separated. The change in the UV absorption gives an idea about the degree of denaturation and renaturation.
Q7 Why denatured DNA not destroyed?
Answer: Denaturation of DNA happens because of the breakage of non covalent interactions thus it is only denatured not destroyed. Polynucleotide strands are made up of sugar phosphate backbone is made up of covalent linkages (bonds) which do not break during the denaturation process.
Q8 Is DNA denaturation reversible?
Answer: Yes, DNA denaturation process is reversible. When the temperature gets lower, the separated strands of DNA starts to reanneal due to the formation of hydrogen bonds between the complementary sequence of DNA. Reannealing of DNA promotes the restoration of DNA double helical structure and hence DNA denaturation is a reversible process.
Q9 Why is it easier to denature DNA than proteins?
Answer: Both DNA and proteins can be denatured by using denaturants like heat, extreme pH, High salt concentration and the presence of other chemical denaturants. Denaturation of DNA is easy to understand while denaturation of proteins is a complex phenomenon because structure of proteins are stabilized by covalent as well as non covalent interactions. Some proteins may denature very easily while some proteins denature at very extreme conditions.
Q10 If DNA is not a protein why do we say that DNA could be denatured?
Answer: A biomacromolecule is said to be denatured when it start to lose its structural integrity or when it changes structure. Since DNA also has a well defined structure, thus when the structure of DNA changes we can say that DNA is being denatured.
Q11 Why are controls used during electrophoresis?
Answer: Controls (positive and negative) are often used when we perform the electrophoresis of DNA. We use control to check the status of the structure of the sample DNA (whether it is double stranded, single stranded, partially double stranded, nicked etc.). Double stranded DNA runs faster in the agarose gel as compared to the single stranded DNA while nicked DNA runs slowest.
Q12 What happens when DNA is damaged?
Answer: The damaged or nicked DNA runs slowest, lags behind and has low mobility. Thus nicked or damaged DNA covers short distances in the agarose gel electrophoresis.
Q13 Which bonds break first when the denaturation of proteins occur?
Answer: Hydrogen bonds along with other non-covalent interactions (hydrophobic, vander waal forces) are disturbed first while covalent interactions like disulphide linkages are broken in the last during the denaturation process.
Q14 How does DNA get transferred from heat killed S strain to rough R strain considering the fact that the DNA can undergo denaturation?
Answer: During heat killing of the virulent S strain of Pnuemococcus its DNA gets denatured but, when it is mixed with the R strain, the temperature is not that high. So, DNA might reannealed during the mixing process and restored its functionality.
Q15 Can denaturing proteins mean breaking them into more fundamental yet functional detectable parts?
Answer: No, Denaturation is only aimed to change the structure of biomacromolecules. Change in structure brings about a change or loss in function of these molecules. Denaturation doesn’t mean destroying or breaking into fundamental units. Denaturation is not digestion.
Q16 What is the difference between thermally stable DNA polymerase and normal DNA polymerases?
Answer: Thermo stable DNA polymerase is obtained from an extremophile bacterium Thermus aquaticus, it is different from the normal DNA polymerase because it can perform replication of DNA even at a temperature above 90oC.
Q17 Why does the denaturation of longer DNA helices show more cooperativity than the denaturation of short strands?
Answer: Since the larger DNA strands has more nucleotides in them, there are more chances of forming complementary base pairs, there are more chances of formation of large number of hydrogen bonds. Therefore, larger DNA fragments show more cooperativity.
Q18 Why does NaOH denature DNA?
Answer: The introduction of NaOH increases the pH of the medium and makes it alkaline, OH ions of released from NaOH interacts with Guanine and Thymine and promotes the breaking of hydrogen bonds which ultimately leads to denaturation of DNA.
Q19 Can the denaturalization of DNA cause by formaldehyde affect DNA fingerprinting?
Answer: No, Formaldehyde won’t effect the outcome of DNA fingerprinting. Since DNA fingerprinting requires restriction digestion and restriction enzymes recognize specific sequences in the double stranded DNA. Formaldehyde may denature DNA but after the removal of formaldehyde (as it is used only in the fixation process after electrophoresis) DNA reverts back to its original conformation thus, DNA fingerprinting is unaffected.
Q20 Is there any instrument available that can distinguish denatured DNA from normal DNA other than doing gel electrophoresis?
Answer: Yes there are some tools and instruments which works on the principle of spectroscopy for the determination of melting temperature and from that we can distinguish between denatured and normal DNA. Other technique is agarose gel electrophoresis of DNA samples.
Q21 What is the effect on denaturation of DNA if Tm value is increased relation between Tm value and denaturation of DNA?
Answer: The higher Tm value indicates that the DNA structure has more stability in comparison with the DNA having lower Tm value.
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I am Abdullah Arsalan , Completed my PhD in Biotechnology. I have 7 years of research experience. I have published 6 papers so far in the journals of international repute with an average impact factor of 4.5 and few more are in consideration. I have presented research papers in various national and international conferences. My subject area of interest is biotechnology and biochemistry with special emphasis on Protein chemistry, enzymology, immunology, biophysical techniques and molecular biology.
