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At a wavelength of 260 nm, the average extinction coefficient for double-stranded DNA is 0.020 (μg/mL) −1 cm −1, for single-stranded DNA it is 0.027 (μg/mL) −1 cm −1, for single-stranded RNA it is 0.025 (μg/mL) −1 cm −1 and for short single-stranded oligonucleotides it is dependent on the length and base composition.
[5] [6] While double-stranded DNA (dsDNA) structure may not traditionally be considered structure, in the typical sense of alternating segments of single- and double-stranded regions, in reality, dsDNA is not simply a perfectly ordered double helix at every location of its length due to thermal fluctuations in the DNA and alternative structures ...
Several formulas are used to calculate T m values. [10] [11] Some formulas are more accurate in predicting melting temperatures of DNA duplexes. [12] For DNA oligonucleotides, i.e. short sequences of DNA, the thermodynamics of hybridization can be accurately described as a two-state process.
However, instead of simply measuring the percentage of double-stranded DNA versus time, the amount of renaturation is measured relative to a C 0 t value. The C 0 t value is the product of C 0 (the initial concentration of DNA), t (time in seconds), and a constant that depends on the concentration of cations in the buffer.
The method used is usually PCR with double-stranded DNA-binding dyes as reporters and the dye used is usually SYBR Green. The DNA melting temperature is specific to the amplified fragment. The results of this technique are obtained by comparing the dissociation curves of the analysed DNA samples. [11]
The following formula takes into account the most important variables that can affect depth of coverage (N=40DG÷R) where "N" is the number of reads, "D" is the desired depth of coverage, "G" is the size of DNA target in base pair, and "R" is final read length.
HRM analysis is performed on double stranded DNA samples. Typically the user will use polymerase chain reaction (PCR) prior to HRM analysis to amplify the DNA region in which their mutation of interest lies. In the sample tube there are now many copies of the DNA region of interest. This region that is amplified is known as the amplicon.
The double-helix model of DNA structure was first published in the journal Nature by James Watson and Francis Crick in 1953, [6] (X,Y,Z coordinates in 1954 [7]) based on the work of Rosalind Franklin and her student Raymond Gosling, who took the crucial X-ray diffraction image of DNA labeled as "Photo 51", [8] [9] and Maurice Wilkins, Alexander Stokes, and Herbert Wilson, [10] and base-pairing ...