Restriction endonucleases with enzymes

Figure 9.3. Restriction map produced by Webcutter. The partial restriction map shows the nucleotide sequence of human lysozyme gene submitted to Webcutter using options for all restriction endonucleases with recognition sites equal to or greater than six nucleotides long and cutting the sequence 2 6 times (at least 2 times and at most 6 times). The restriction profile (map) is returned if Map of restriction sites is selected for display. The tables by enzyme name and by base pair number can be also returned if displays for Table of sites, sorted alphabetically by enzyme name Table of sites, sorted sequentially by base pair number are chosen.

Restriction endonuclease Enzymes that catalyze the double-strand cleavage of DNA at specific base sequences. Many restriction endonucleases with different sequence specificities have been found in bacteria they are used extensively in molecular genetics. 

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). 

Cutting with restriction endonucleases is very useful for moving specific pieces of DNA around from place to place. It s also a useful way to name pieces of DNA. For example, a piece of DNA that is cut from a bigger piece of DNA is often named by size and given a surname that corresponds to the two restriction enzymes that did the cutting—the 0.3-kb EcoRI-BamHI fragment. Restriction enzymes themselves are named for the bacterial strains from which they were initially isolated. 

BamHI and other restriction endonucleases are dimeric enzymes that bind to a DNA palindrome and cut both strands at equivalent positions. The cut leaves two ends with complementary overhangs that will... 

PUTTING YOUR DNA INTO A VECTOR Vectors are specialized pieces of DNA used to move other pieces of DNA around. Modern vectors are usually either bacterial plasmids or viral genomes. The act of isolating your DNA in the first place usually involves putting it into a vector and then selecting the vector that has your DNA in it. DNA pieces (called inserts when they are placed in a vector) are usually placed in vectors using restriction endonucleases. The vector is cut with two restriction enzymes of different specificity (Fig. 6-3). This removes a... 

This activity is intended to be performed in conjunction with Experiment 66. Restriction endonucleases, or restriction enzymes, cleave DNA at specific base sequences, fragmenting the DNA into smaller pieces. The two strands of a DNA double helix are cleaved at different places, resulting in uneven fragments called sticky ends. Cleavage of DNA by restriction enzymes is a required first step in various types of DNA analysis, including DNA fingerprinting and recombinant DNA technology. 

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. 

The cuts in the two strands are made at the points indicated by the arrows. This one endonuclease will cut almost any DNA into long pieces averaging about 5000 base pairs each. These pieces can in turn be cleaved by other restriction endonucleases to form smaller fragments. Since there are about 2400 of these enzymes known, with 188 different specificities,536 it is possible to cut any piece of DNA down to a size of 100-500 base pairs, ideal for sequencing.537 539 Each fragment has known sequences at the two ends. Some restriction enzymes cleave outside their specific recognition sequence (see Table 26-2). Some recognize 16-nucleotide palindromes and cut at rare sites. 

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