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When an electric field is applied, the DNA will begin to move through the gel, at a speed roughly inversely proportional to the length of the DNA molecule (shorter lengths of DNA travel faster) — this is the basis for size dependent separation in standard electrophoresis. In TGGE there is also a temperature gradient across the gel.
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 ...
Thus the denaturation can occur at the Tc, proceed to primer annealing, and then polymerase-mediated extension. Each round of amplification will include these three stages in that order. By utilizing the lower denaturation temperature, the reaction will discriminate toward the products with the lower Tm – i.e. the variant alleles.
In biochemistry, denaturation is a process in which proteins or nucleic acids lose folded structure present in their native state due to various factors, including application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), agitation and radiation, or heat. [3]
The polymerase chain reaction is the most widely used method for in vitro DNA amplification for purposes of molecular biology and biomedical research. [1] This process involves the separation of the double-stranded DNA in high heat into single strands (the denaturation step, typically achieved at 95–97 °C), annealing of the primers to the single stranded DNA (the annealing step) and copying ...
While human polymerase optimally works at 37 °C (99 °F), DNA does not denature until 94–98 °C (201–208 °F). [20] This poses a problem as at these temperatures the human DNA polymerase would denature during the denaturation step of the PCR reaction resulting in a non-functioning polymerase protein and a failed PCR.
The AP endonucleases also participate in 3' end processing. Besides opening AP sites, they possess 3' phosphodiesterase activity and can remove a variety of 3' lesions including phosphates, phosphoglycolates, and aldehydes. 3'-Processing must occur before DNA synthesis can initiate because DNA polymerases require a 3' hydroxyl to extend from.
Afterwards, the 3’ ssDNA invades the template DNA, and displaces a DNA strand to form a D-loop. DNA polymerase and other accessory factors follows by replacing the missing DNA via DNA synthesis. Ligase then attaches the DNA strand break, [10] resulting in the formation of 2 Holliday junctions. The recombined DNA strands then undergoes ...