DsDNA denaturation

Fig. 30.3. Evaluation of the efficiency of the dsDNA denaturing treatment. (A) Heating treatment of the dsDNA before immobilization. (B) Heating treatment of the dsDNA before immobilization plus denaturing alkaline treatment after immobilization. 153.8 ng of poly(dA)poly(dT) as DNA target 9.97 pmol dT(50)-biotin and 9.00 /ig of enzyme conjugate. The nonspecific adsorption signal is shown in black (more details in Ref. [2]).

The stability of dsDNA can be determined through a temperature denaturation study. Native dsDNA will denature to ssDNA as the temperature of the solution is increased. This is a reversible process, and the ssDNA will anneal to dsDNA on slowly cooling the denatured solution. This behavior can be followed spectroscopically (Fig. 6.11) by noting the increase in A260nm temperature, indicating the hyperchromic shift as the dsDNA denatures to ssDNA. The... 

As further corroboration of the dsDNA denaturation in poor solvents, force measurements were performed in a denaturing environment, an aqueous solution... 

We have used cesium chloride density gradient centrifugation to clean up phaiol-chloroform extracted, ethanol precipitated DNA and measured the "hyperchromic shift" when dsDNA denatures to ssDNA as an indication of DNA purity. A "hyperchromic shift" of more than 40% indicates that the DNA is of sufficient purity for quantitative hybridization. 

Unwinding (denaturation) of dsDNA to provide an ssDNA template. 

Dissolve the DNA sample to be modified at a concentration of 20-100 pg/ml in 10 mM Tris, 1 mM EDTA, pH 7.4. Note The sample may be heated to denature and solubilize genomic DNA and then cooled to form dsDNA for modification. 

This conclusion is also supported by the fact that, in contrast to ssDNA, the oxidation signal coming from dsDNA is poorly developed at both GC and GC(ox). This is probably attributable to the electroactive A and G residues in dsDNA being inaccessible to the surface, while most bases in denatured DNA can freely interact with the GC(ox) surface. On the other hand, the hydrogen-bonded bases in native DNA are hidden within the double heUx, a serious steric barrier to electron transfer between the purine and the GC(ox). 

Electrochemical impedance measurements of the physical adsorption of ssDNA and dsDNA yields useful information about the kinetics and mobihty of the adsorption process. Physical adsorption of DNA is a simple and inexpensive method of immobilization. The ability to detect differences between ssDNA and dsDNA by impedance could be applicable to DNA biosensor technology. EIS measurements were made of the electrical double layer of a hanging drop mercury electrode for both ssDNA and dsDNA [34]. The impedance profiles were modeled by the Debye equivalent circuit for the adsorption and desorption of both ssDNA and dsDNA. Desorption of denatured ssDNA demonstrated greater dielectric loss than desorption of dsDNA. The greater flexibility of the ssDNA compared to dsDNA was proposed to account for this difference. 

Evidence of the electrical conductivity of DNA and of its important mechanisms has been discussed for a long time and has led to a theory of electron conduction in biopolymers [25, 82]. From this it appeared that the major carrier of conductivity is either electronic or ionic, depending on the temperature of the sample, the water content, and the fact that the conductivity of native samples is higher than that of denatured samples. Following electrochemical oxidation of dsDNA and ssDNA in electrolyte solutions over a wide range of pH, interesting electrochemical properties of a glassy carbon electrode with dsDNA or ssDNA adsorbed on the electrode surface were observed [68]. 

Fig. 1. Preparation of ssDNA from biotinylated PCR-amplified DNA. (a) DNA is amplified using one primer that has a biotin molecule (B) at the 5 end and one primer that lacks biotin, (b) Double-stranded amplified DNA is captured on a streptavidin-coated magnetic bead via the biotin molecule, (c) The dsDNA is denatured, with the biotinylated strand remaining attached to the streptavidin-coated magnetic bead and the nonbiotinylated strand being released into the supernatant, (d) The nonbiotinylated strand is used as an ssDNA sequencing template.

The dideoxy DNA sequencing method begins with the denaturation of double-stranded DNA (dsDNA) into single-stranded DNA. The ssDNA is then annealed with a fluorescent dye-labeled primer, which is an oligodeoxynucleotide 20 bases long. The heteroduplex formed is then incubated in four separate reactions. 

A single DNA molecule can be amplified millions of times by replicating a three-step cycle. In the first step the dsDNA sample is denatured by heating to 95°C. In step 2 the temperature is quickly lowered to 50°C and an oligonucleotide primer is added. The primer hybridizes to complementary sequences on the ends of the two strands. During step 3, DNA synthesis occurs as the temperature is raised to 70°C, the optimal temperature of Taq polymerase. The cycle is then repeated with both old and new strands serving as templates. 

When testing DNA amphfied samples, the amplicons were denatured to dissociate the dsDNA into ssDNA, using the approaches reported in Sect. 2.3 of this chapter. In Fig. 5 the response obtained with the different denaturation procedures using the biotinylated probe and streptavidin-modified surfaces is shown. A good reproducibility was found with PCR-amplified material (CV% 6 ( = 3)). 

Amplicons of double-stranded DNA (dsDNA) are melted by application of heat and denaturating chemicals such as formamide and NaOH. 

Step 1 - DNA denaturation The temperature of the reaction mixture is elevated to 94-95 °C. The double stranded sample DNA (dsDNA) is denatured into single strands (ssDNA), as the hydrogen bonds between the complementary bases are broken. The polymerase enzyme is inactive at this temperature. 

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