Endonuclease, restriction

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. 

Cutting DNA at precise locations. Sequence-specific endonucleases (restriction endonucleases) provide the necessary molecular scissors. 

One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecules involved. The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded DNA into smaller, more manageable fragments, has opened the way for DNA analysis. Because each enzyme cleaves DNA at a specific nucleotide sequence, restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments. 

A cassette-replacement approach was used to facilitate the introduction of amino acid mutations at various sites of the thrombin receptor. First, unique endonuclease restriction enzyme sites were generated at several positions within the thrombin receptor cDNA by mutating the nucleotide sequences. Second, the polymerase chain reaction (PCR) with primers encoding for the desired mutations was used to generate the cDNA cassette with the appropriate endonuclease restriction enzyme sites for replacement of the wild-type sequence. The locations for the introduction of the sites were chosen based on two requirements. They needed to be at or near regions of the cDNA sequence that codes for amino acids at junctions of transmembrane domains and extracellular loops. Also, introduction of the sites did not alter the amino acid sequence of the protein. The site-directed mutagenesis method of Kunkel et al.28 was used to introduce the mutations required for generating the... 

Cutting DNA at precise locations to yield the DNA segment (gene) of interest by use of sequence specific restriction endonuclease (restriction enzyme). 

More than 100 restriction endonucleases (restriction enzymes) have been isolated and purified from various bacterial species. All but a few of these enzymes recog-... 

DNA sequencing Maxam-Gilbert method restriction endonuclease restriction fragment palindrome Sanger dideoxy method DNA synthesis DMT ether phosphoramidite phosphite polymerase chain reaction (PCR)... 

The restriction nucleolysis of DNA by restriction endonucleases (restriction enzymes), which recognizes specific sequences on the target DNA will be described in the Chapter 13. 

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