DNA, denaturation

The double-stranded DNA to be amplified is heated in the presence of Taq polymerase, Mg2+ ion. the four deoxynucleotide triphosphate monomers (dNTPs), and a large excess of two short oligonucleotide primers of about 20 bases each. Each primer is complementary to the sequence at the end of one of the target DNA segments. At a temperature of 95 °C, double-stranded DNA denatures, spontaneously breaking apart into two single strands. 

In contrast to the reactivity of N-sulfonyloxy and N-acetoxy esters of arylamides and arylamines, the relative reactivity of protonated N-hydroxy arylamines with nucleophiles generally decreases in the order DNA > denatured DNA > rRNA = protein > tRNA nucleotides s nucleosides s methionine = GSH (2,13-17,30,36,40,127,129, 130). Furthermore, the rate of reaction with DNA was found to be not only first order with respect to N-hydroxy arylamine concentration, but also first order with respect to DNA concentration (127,129,131). These data suggested that the reaction mechanism was... 

The double helix can be denatured by heating (melting). Denatured DNA, like denatured protein, loses its structure, and the two strands separate. Melting of DNA is accompanied by an increase in the absorbance of UV light with a wavelength of 260 nm. This is termed hyperchromicity and can by used to observe DNA denaturation. DNA denaturation is reversible. When cooled under appropriate conditions, the two strands find each other, pair correctly, and reform the double helix. This is termed annealing. 

Diagrammatic representation of DNA denaturation. Upon perturbation, the first "melting" away of structure occurs within AT-rich regions, and further perturbation causes this local disorder to grow, such that GC-rich regions are the last to become disordered. The DNA unwinding reaction is limited by viscous resistance. 

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

The first step of a PCR involves DNA denaturation at 90-95 °C, in a buffered, neutral, aqueous solution containing DNA polymerase, the four deoxynucleotide triphosphates and Mg++, in the presence of a large excess of the two primers (Fig. 27). In the second step, the temperature of the reaction is lowered to about 10 °C below the melting temperature of the primers and the primers (which are considerably smaller than the DNA) are allowed to hybridize to their complementary sequence on the DNA template molecule. This temperature is still too high for the DNA to fully renature. The temperature is then raised to 72 °C, the optimal temperature for extension of the primers by the DNA polymerase, which catalyses the addition of nucleotide triphosphates to extend the sequence in each direction from the... 

Duplex Structures Can Form Supercoils DNA Denaturation Involves Separation of Complementary Strands... 

The graph shows E. coli labeled with radioactive thymidine for a short pulse (10 s) followed by a chase with an excess of nonradioactive thymidine. The DNA is then extracted and centrifuged in alkaline sucrose gradients (under high pH conditions the DNA denatures). Explain what these data imply, and interpret these results in light of our current model for DNA replication. 

Figure 6.3. Effect of heat on DNA. At high temperature and low ionic strength, the two strands of DNA at A-T-rich regions fall apart, first forming bubble structures along the length of the DNA. As the temperature increases, the size of the bubble increases and the G-C regions also fall apart. Extreme pH ranges also cause DNA denaturation. (Reproduced from Textbook of Biochemistry with Clinical Correlations, T. M. Devlin, ed., Wiley, New York, 1982.)...

Double-stranded DNA denatures into single strands as the temperature rises but renatures into a double-stranded structure as the temperature falls. Any two single-stranded nucleic acid molecules can form double-stranded structures (hybridize) provided that they have sufficient complementary nucleotide sequence to make the resulting hybrid stable under the reaction conditions. 

Which will have a greater surface of interaction—DNA in a double helix or the same DNA denatured, single-stranded random coil Justify your answer with a diagram of helix-to-coil transition. 

DNA is stable at high temperatures up to 60-65°C. Raising the temperature induces DNA denaturation, and the two-single strands split up. This can be evidenced by following the OD variation of DNA at 260 nm at increasing temperatures from 20 to 82°C (Marmur and Doty 1959) (see also Figure 12.14). 

We suggest here following DNA denaturation by following the fluorescence emission and variation of excitation spectra of the ethidium bromide-DNA complex at increasing temperatures from 50 to 85°C. This experiment should be done at the end of a titration experiment when you have complete binding of ethidium bromide within DNA strands. Figure 12.15 shows clearly that the fluorescence intensities of both excitation and emission spectra decrease when the temperature increases. It is important to indicate that free ethidium bromide in solution does not show any significant fluorescence. 

Plotting the normalized fluorescence intensities of excitation and emission spectra vs temperature yields a sigmoidal plot (Figure 12.16) with an inflection point equal to that observed when thermal DNA denaturation is observed by following the ODs at 260 nm (Figure 12.14). 

In cases where targets of different abundance are to be stained, a method must be selected to best balance the signals. Combining ISH and IHC on one slide is particularly challenging because targets require very different pre-treatment protocols. Since ISH processes such as DNA denaturing are not compatible with the presence of the antibodies for IHC, the ISH protocol is normally performed first. 

Understand the process of DNA denaturation and annealing and the information that can be gleaned from such parameters as Tm and C0f. Know how the physical parameters of DNA change as it is denatured. 

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