A simple, general procedure for purifying restriction endonucleases.
نویسندگان
چکیده
A simple, general method for purifying restriction endonucleases is described. The method employs precipitation of nucleic acids from crude extracts with polyethyleneimine followed by affinity chromatography on columns of heparin covalently linked to agarose. Most of the sixteen enzymes tested could be purified to a degree sufficient for DNA sequencing work by this method sometimes supplemented by at most one step of ion exchange chromatography.
منابع مشابه
PCR–RFLP of isolated Salmonella from poultry with Sau3AI and HhaI restriction endonucleases in Arak
Salmonella enterica is considered as one of the major pathogens in public health, worldwide. Diseases caused by Salmonella enterica serovars are especially prevalent in developing countries. It is one of the emerging pathogen in food-borne diseases, which is often found in contaminated chicken eggs. The aim of this study was to apply a PCR–RFLP method using Sau3AI and HhaI restriction endonucle...
متن کاملA rapid and simple method for detection of type II restriction endonucleases in cells of bacteria with high activity of nonspecific nucleases.
In this work we describe a novel, rapid and simple microscale procedure for identification of restriction endonuclease activity in bacteria lysates, which contain high levels of non-specific DNA nucleases.
متن کاملMassively parallel characterization of restriction endonucleases
Restriction endonucleases are highly specific in recognizing the particular DNA sequence they act on. However, their activity is affected by sequence context, enzyme concentration and buffer composition. Changes in these factors may lead to either ineffective cleavage at the cognate restriction site or relaxed specificity allowing cleavage of degenerate 'star' sites. Additionally, uncharacteriz...
متن کاملSequence determination of restriction-endonuclease recognition sites.
the enzyme DpnII, produced by another strain of D. pneumoniae, recognizes the same sequence, but is inhibited by this same methylated base. The significance of the occurrence of modified nucleotides in eukaryotic DNA is not as yet understood, although a relationship between modification and gene expression has been proposed by several workers (Waalwijk & Flavell, 1978; Bird et al., 1979). By us...
متن کاملEnzymatic Cleavage of Type II Restriction Endonucleases on the 2′-O-Methyl Nucleotide and Phosphorothioate Substituted DNA
The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI) were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN) and phosphorothioate (PS). Substitutions were made in the recog...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Nucleic acids research
دوره 4 8 شماره
صفحات -
تاریخ انتشار 1977