Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new...

The increasing interest in methylation patterns of mammalian DNA demands a simple and reliable method to clearly differentiate between cytosine (C) and 5-methylcytosine (5-MeC) within a specific DNA region. The simplest and most commonly used approach is the analysis with restriction endonucleases exhibiting different methylation sensitivities, like the MspI/HpaII pair of isoschizomeric enzymes...

Restriction endonucleases recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. The first method presented in this unit is the cleavage of a single DNA sample with a single restriction endonuclease. A...

The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show...

A computer program is described which allows for the manipulation of restriction maps of various DNA fragments to demonstrate techniques used in DNA cloning and to predict and/or confirm experimental results. This program is capable of reading in restriction enzyme cleavage sites for several different DNA molecules of interest. This information is then compiled in order to form restriction maps...