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After introducing a nick in the system, the negative supercoil gradually unwinds (c) until it reaches its final, circular, plasmid state (d). [2] Nicked DNA can be the result of DNA damage or purposeful, regulated biomolecular reactions carried out in the cell. During processing, DNA can be nicked by physical shearing, over-drying, or enzymes.
As a summary, a typical DNA rolling circle replication has five steps: [2] Circular dsDNA will be "nicked". The 3' end is elongated using "unnicked" DNA as leading strand (template); 5' end is displaced. Displaced DNA is a lagging strand and is made double stranded via a series of Okazaki fragments. Replication of both "unnicked" and displaced ...
In the case of large DNA molecules, the DNA is frequently cut into smaller fragments using a DNA restriction endonuclease (or restriction enzyme). In other instances, such as PCR amplified samples, enzymes present in the sample that might affect the separation of the molecules are removed through various means before analysis. Once the nucleic ...
An example of these techniques is the "Quikchange" method, [16] wherein a pair of complementary mutagenic primers are used to amplify the entire plasmid in a thermocycling reaction using a high-fidelity non-strand-displacing DNA polymerase such as Pfu polymerase. The reaction generates a nicked, circular DNA.
Nicked open-circular DNA has one strand cut. Relaxed circular DNA is fully intact with both strands uncut but has been enzymatically relaxed (supercoils removed). This can be modeled by letting a twisted extension cord unwind and relax and then plugging it into itself. Linear DNA has free ends, either because both strands have been cut or ...
A nicking enzyme (or nicking endonuclease) is an enzyme that cuts only one strand of a double-stranded DNA or RNA molecule [1] at a specific recognition nucleotide sequence known as the restriction site. Such enzymes hydrolyze (cut) only one strand of the DNA duplex, to produce DNA molecules that are “nicked”, rather than cleaved. [2] [3]
Estimation of the DNA concentration by comparing the intensity of the nucleic acid band with the corresponding band of the size marker. [34] Analysis of products of a polymerase chain reaction (PCR), e.g., in molecular genetic diagnosis or genetic fingerprinting; Separation of DNA fragments for extraction and purification.
The reference state (or parameter) L 0 of a circular DNA duplex is its relaxed state. In this state, its writhe W = 0. Since L = T + W, in a relaxed state T = L. Thus, if we have a 400 bp relaxed circular DNA duplex, L ~ 40 (assuming ~10 bp per turn in B-DNA). Then T ~ 40. Positively supercoiling: