Restriction endonuclease enzymes

Restriction enzymes Endonucleases isolated from bacteria that will selectively cleave DNA having specific nucleotide sequences. The enzyme recognizes particular palindromic nucleotide sequences and will hydrolyze both strands within that sequence. Molecular biologists use these enzymes in various recombinant DNA techniques. 

Restriction enzymes (endonucleases) typically cut DNA at specific 4- to 8-bp palindromic sequences, producing defined fragments that often have self-complementary single-stranded tails (sticky ends). 

The enzymes of most economical importance, such as amylases, glucose isomerase, and proteases, will be discussed individually. Altogether they account for almost 90 percent of the total sales of enzymes. Other industrially important enzymes with relative small sales volumes are listed in Table 24.6, along with their microbial sources and commercial applications. In addition, there are many microbial enzymes that are used for analytical, clinical, and research purposes they include hexokinases, pyruvate kinase, uricase, glucose-6-phosphate dehydrogenase, amino acid oxidase, aminopeptidase, and others. Restriction enzymes, endonucleases, have been used widely in recombinant DNA research. Glyco-syltransferases have received much attention recently as glycobiology research picks up steam. 

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. 

The detection of restriction fi agment length polymorphisms (RFLPs) facilitates prenatal detection of hereditary disorders such as sickle cell trait, beta-thalassemia, infant phenylketonuria, and Huntington s disease. Detection of RFLPs involves cleavage of double-stranded DNA by restriction endonucleases, which can detect subtle alterations in DNA that affect their recognized sites. Chapter 40 provides further details concerning the use of PCR and restriction enzymes for diagnosis. 

Restriction enzymes are sequence-specific endonucleases that cut double-stranded DNA at specific sites. 

Most useful restriction enzymes cut DNA at specific recognition sites, usually four to six nucleotides in length. There can be multiple restriction sites for a single endonuclease within a given piece of DNA, there can be only one (a unique restriction site), or there can be none. It all depends on the sequence of the specific piece of DNA in question. 

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. 

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. 

Bacteria contain enzymes that catalyse the breaking of phosphodiester links between nucleotides in DNA at specific sites, to which the enzyme is directed by a short sequence of bases. These are known as restriction enzymes and they have resulted in remarkable progress in analysing sequences of DNA fragments. They are endonucleases, i.e., they cleave DNA at the phosphodiester bonds within, rather than at the ends, of DNA chains. They cleave bonds such that sequences of nucleotides, typically 4-8 base pairs, are produced. These are the restriction sequences. 

DNA molecules, whether linear or circular, can be cut by means of enzymes known as restriction enzymes. A restriction enzyme is an enzyme that recognizes certain sequences of nitrogen bases and breaks the bonds at some point within that sequence. Since these enzymes work on bonds within the DNA molecule, they are sometimes called restriction endonucleases. 

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. 

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