Precise DNA manipulation is a key enabling technology for synthetic biology. Approaches based on restriction digestion are often limited by the presence of certain restriction enzyme recognition sites. Recent development of restriction-free cloning approaches has greatly enhanced the flexibility and speed of molecular cloning. Most restriction-free cloning methods focus on DNA assembly. Much le...

One restriction enzyme map of Staphylococcus aureus bacteriophage phi 11 DNA was established by reciprocal double digestions with the enzymes EcoRI, HaeII, and KpnI. The sequential order of the EcoRI fragments was thereafter established by a novel approach involving blotting of DNA partially cleaved with EcoRI and the probing the blots with nick-translated terminal fragments. A circular map of ...

A method is described which allows the preparation of reproducible partial digests without previous establishment of the incubation conditions. It is based on a combined application of dam methylase and the restriction endonuclease Mbol, both recognizing the sequence 5'-GATC-3' but Mbol unable to cut the methylated site. Due to their competition for the same substrate the DNA is partially diges...

All restriction enzymes examined are phosphodiesterases generating 3'-OH and 5'-P ends, but one restriction enzyme (restriction glycosylase) excises unmethylated bases from its recognition sequence. Whether its restriction activity involves endonucleolytic cleavage remains unclear. One report on this enzyme, R.PabI from a hyperthermophile, ascribed the breakage to high temperature while another...

Requirement of divalent cations for DNA cleavage is a general feature of type II restriction enzymes with the exception of few members of this group. A new type II restriction endonuclease has been partially purified from Lactococcus lactis KLDS4. The enzyme was denoted as LlaKI and showed to recognize and cleave the same site as FokI. The enzyme displayed a denatured molecular weight of 50 kDa...

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage A that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, b...

Restriction enzymes are well known as reagents widely used by molecular biologists for genetic manipulation and analysis, but these reagents represent only one class (type II) of a wider range of enzymes that recognize specific nucleotide sequences in DNA molecules and detect the provenance of the DNA on the basis of specific modifications to their target sequence. Type I restriction and modifi...

The DNAs of a varicella-zoster virus vaccine and its parental virus were compared by CsCl buoyant density centrifugation and restriction enzyme cleavage analysis. The varicella-zoster virus vaccine DNA showed a heterogeneous buoyant profile and altered restriction enzyme cleavage patterns. These changed properties are probably the result of the accumulation of virus containing defective varicel...

It is widely recognized that the cleaving rate of a restriction enzyme on target DNA sequences is several orders-of-magnitude faster than the maximal one calculated from the diffusion-limited theory. It was therefore commonly assumed that the target site interaction of a restriction enzyme with DNA has to occur via two steps: one-dimensional diffusion along a DNA segment, and long-range jumps c...

Biological assays at the single molecule level are crucial to fundamental studies of DNA-protein mechanisms. In order to cater for high throughput applications, one area of immense research potential is single-molecule bioassays where miniaturized devices are developed to perform rapid and effective biological reactions and analyses. With the success of various emerging technologies for enginee...