Restriction endonucleases commercially available

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

Exonucleases. Like the endonucleases they are restriction enzymes which act at the 3 or 5 ends of linear DNA by hydrolysing off the nucleotides. Although they are highly specific for hydrolysing nucleotides at the 3 or 5 ends of linear DNA, the number of nucleotides cleaved are time dependent and usually have to be estimated from the time allocated for cleavage. Commercially available exonucleases are used without further purification. 

The first step in DNA sequencing is to cleave the enormous chain at known points to produce smaller, more manageable pieces, a task accomplished by the use of restriction endonucleases. Each different restriction enzyme, of which more than 3500 are known and approximately 200 are commercially available, cleaves a DNA molecule at a point in the chain where a specific base sequence occurs. For example, the restriction enzyme Alul cleaves between G and C in the four-base sequence AG-CT. Note that the sequence is a palindrome, meaning that the sequence (5 )-AGCT-(3 ) is the same as its complement (3 )-TCGA-(5 ) when both are read in the same 5 — 3 direction. The same is true for other restriction endonucleases. 

These enzymes are isolated from bacteria, their natural source. There are many different restriction endonucleases isolated from a variety of bacteria that are now readily available commercially. In bacteria they act as part of a restriction/modification system that protects the bacteria from infection by DNA viruses. 

Double stranded DNA can be enzymatically cleaved by commercially available endonucleases (restriction enzymes). These enzymes recognise specific, usually palindromic, sequences and cut specifically at those positions. 

Restriction endonucleases are bacterial enzymes that cleave DNA at sequence specific sites. They were first discovered in 1970 [19]. Almost 2000 restriction enzymes have been identified since, and several hundred of these are commercially available [1]. Many mutations remove or create a particular restriction site in the DNA sequence. These mutations can be identified by PCR amplification, incubation of the product with the appropriate enzyme followed by visualisation of the fragments on an agarose gel. 

Type II restriction endonucleases recognize specific base sequences in double-stranded DNA, and cleave both strands of the duplex. More than 600 restriction endonucleases have been reported in the literature. Some 121 restriction endonucleases are listed in Current Protocols in Molecular Biology as commercially available ( 3). Table II, adapted from the 1988 Sigma Chemical Company Catalog, lists 35 of the endonucleases available from that source. Endonucleases are found in many bacteria where their function is to hydrolyze foreign DNA introduced by phage infection, conjugation or transformation. The first restriction endonucleases were discovered and isolated in 1968 (4,5). 

Table V-4 Some Commercially Available Type II Restriction Endonucleases...

The power aind utility of recombinant DNA methods, which include techniques of nucleic acid hybridization, are based on the ability to manipulate DNA in defined ways. These molecular tools are dependent on the commercial availability of a variety of well-characterized enzymes for DNA and RNA modification restriction endonucleases which recognize and cleave specific DNA sequences to create DNA fragments of unique size, DNA and RNA polymerases. 

The most often used restriction endonucleases for staphylococci are HinAlll, Clal (Tenover et al. 1994), EcoRI (Blumberg et al. 1992), EcoRV or Kpnl (Blanc et al. 1994). The fragments containing specific sequences are then detected using a labelled piece of homologous DNA as a probe. The most often used is DNA complementary to 16S and 23S rRNA isolated from Escherichia coli and commercially available for ribotyping (Falkinham 1994 Pfaller and Hollis 2004). 

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