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Spontaneous deamination of 5-methylcytosine results in thymine and ammonia. This is the most common single nucleotide mutation. In DNA, this reaction, if detected prior to passage of the replication fork, can be corrected by the enzyme thymine-DNA glycosylase, which removes the thymine base in a G/T mismatch. This leaves an abasic site that is ...
DNA denaturation occurs when hydrogen bonds between base pairs are disturbed. The non-covalent interactions between antiparallel strands in DNA can be broken in order to "open" the double helix when biologically important mechanisms such as DNA replication, transcription, DNA repair or protein binding are set to occur. [19]
Methylation often occurs to arginine or lysine and involves adding a methyl group to a nitrogen (replacing a hydrogen). The R groups on these amino acids can be methylated multiple times as long as the bonds to nitrogen does not exceed 4. Methylation reduces the ability of these amino acids to form hydrogen bonds so arginine and lysine that are ...
In the less extensive technique of equilibrium unfolding, the fractions of folded and unfolded molecules (denoted as and , respectively) are measured as the solution conditions are gradually changed from those favoring the native state to those favoring the unfolded state, e.g., by adding a denaturant such as guanidinium hydrochloride or urea.
Heat denaturation of DNA, also called melting, causes the double helix structure to unwind to form single stranded DNA. When DNA in solution is heated above its melting temperature (usually more than 80 °C), the double-stranded DNA unwinds to form single-stranded DNA. The bases become unstacked and can thus absorb more light.
DNA polymerase, the main enzyme to catalyze the polymerization of free deoxyribonucleotides into a newly forming DNA strand, plays a significant role in the occurrence of this mutation. When DNA polymerase encounters a direct repeat, it can undergo a replication slippage. [4] Strand slippage may also occur during the DNA synthesis step of DNA ...
Two-step RT-PCR, as the name implies, occurs in two steps. First the reverse transcription and then the PCR. This method is more sensitive than the one-step method. Kits are also useful for two-step RT-PCR. Just as for one-step PCR, use only intact, high-quality RNA for the best results. The primer for two-step PCR does not have to be sequence ...
Denaturation: If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged thymine residues of DNA to a positively charged amino groups of membrane ...