Homing Endonucleases and Inteins: 16 (Nucleic Acids and Molecular Biology)
For a 3, base pair plasmid with a single recognition site, there are 0. The ability of a restriction enzyme to find a single site by linear diffusion in the supercoiled plasmid is also presumed to be different than for any of the sites on a linear substrate. Although it is not common, some enzymes exhibit differences in their ability to cut supercoiled DNA depending on the buffer conditions used.
For example, Sac II exhibits a pronounced difference in its ability to cut supercoiled plasmids depending on buffer conditions, but this sensitivity is not seen nearly as dramatically with linear substrates. Table 2. In order to recognize and cleave their recognition sequence, most restriction enzymes need some flanking DNA. Because of this it can be difficult to achieve complete digestion of PCR products that have restriction sites engineered near the end of a primer or to perform double digests using two enzymes that cut at sites close to each other in a polylinker region.
Such digestions may be improved by using long hour incubation times. When performing multiple digests within a polylinker region, it is important to determine if the sites overlap such that cleavage at one site will destroy another. Alternatively, if the DNA is first digested with SmaI, it will leave the sequence shown below, which can be digested with Kpn I, although there may be problems due to a lack of flanking bases.
This data can be used to help determine the order in which two enzymes should be used for the most efficient multiple digests, or to predict whether enzymes will work effectively in a double-digest. In general, the addition of extra bases upstream of an engineered restriction site in a PCR primer will greatly increase the efficiency of digestion of the amplification product, but this is dependent on the enzyme used. PCR products in which the first base pair of the restriction site was flush with 0 , or 1, 2, or 3 base pairs away from the end of the fragment were tested with a variety of enzymes.
Purified PCR fragments ng were digested at least twice with 0. The addition of upstream bases to PCR primers is not the only method used to improve digestion efficiency. A number of protocols have been proposed to improve digestion including proteinase K treatment to remove any thermostable polymerase that may be blocking the DNA, end-polishing with Klenow or T4 DNA Polymerase and the addition of spermidine.http://outer-edge-design.com/components/mspy/3782-mobile-hangouts.php
Home and away- the evolutionary dynamics of homing endonucleases
However, none of these methods have been shown to improve cloning efficiency significantly 4 5. An additional drawback to the incorporation of restriction enzyme sites in PCR primers is that it can be quite difficult to resolve digested PCR products from those that remain uncut. This allows identification of products that have been cut successfully because the label is lost upon digestion 6.
An alternative method that has been used successfully to improve digestion of PCR products is to concatamerize the fragments after amplification 1 5. This is achieved by first treating the cleaned up PCR products with T4 Polynucleotide Kinase if the primers have not already been phosphorylated. The ends will already be blunt if a proofreading thermostable polymerase such as Pfu was used or may be treated with T4 DNA Polymerase to polish the ends if a non-proofreading polymerase such as Taq was used.
This effectively moves the restriction enzyme sites away from the ends of the fragments and allows efficient digestion. This troubleshooting guide addresses common problems that may be encountered while using restriction enzymes. If problems persist contact Promega Technical Services at techserv promega.
Benutzerkonto anlegen. Contact Customer Service Kennwort vergessen? Bitte kontaktieren Sie den Kundenservice, um Ihr Benutzerkonto zu entsperren. Benutzername Username not found. Aktuelles Passwort Falsches Kennwort. Das Kennwort entspricht nicht den Richtlinien.
Marlene Belfort - Publications
Das Kennwort wurde bereits verwendet. There was an issue logging into your account. Please try again or contact Customer Service. Name This field is required. Email Address Please enter a valid email address. Der Benutzername existiert bereits. Haben Sie ein Benutzerkonto?
Sie haben ein Promega. Laut unseren Aufzeichnungen wurde die E-Mailadresse bereits registiert.
Promotions Angebote und Rabatte View all promotions. Beratung anfordern. Support Center erkunden. Ansprechpartner im Vertrieb finden. Ihr Warenkorb Artikel im Warenkorb 0 Warenkorb anzeigen. Zur Kasse. History Restriction enzymes recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to these sequences.
References Roberts, R. Gene , 19— Luria, S. Bertani, G. Arber, W. Host controlled modification of bacteriophage lambda. Dussoix, D. Control over acceptance of DNA from infecting phage lambda.
Linn, S. And Arber, S.
Purification and general properties. USA 59 , Meselson, M. And Yuan, R. Nature , —4. Smith, H. Kelly, T. Back to top. Types, Definitions and Genomic Organization A. Recognition Sequences Most restriction endonucleases recognize palindromic or partially palindromic sites. Types and General Properties of Restriction Endonucleases The table below gives the types and general properties restriction endonucleases. Type I EC 3. References Williams, R. Roberts, R. Kong, H. Sears, L. Nucleic Acids Res. Reuter, M. Oller, A. Szybalski, W. Gene , 13— Belfort, M.