Restriction endonucleases units

Enzymes, specialized proteins, are used as designing tools for genetic engineering. One of these enzyme tools consists of restriction endonucleases that recognize a specific series of base pairs. They split the DNA at these specific points. This splitting is called lysing , which in reality is simply the hydrolysis of DNA units as shown in the following structure ... 

The calibration of the E. coli genetic map in minutes was a temporary expedient. It was followed by physical maps expressed directly as micrometers of DNA length (total length 1.6 mm) or thousands of nucleotide units (kb). A physical map obtained by restriction enzyme mapping is shown in Fig. 26-4B. To obtain this map DNA fragments were prepared using specific restriction endonucleases (Section E, 1). 

The MCS is a stretch of unique restriction endonuclease cleavage sites between the promoter and the poly (A) signal in expression plasmid vectors, and is used as a common area to insert a cDNA or gene of interest. By using the MCS, the genes of interest can be inserted and assayed with ease. Sticky-end and blunt-end vector strands are available at different restriction sites. Although the MCS is very convenient, it should be noted that these sites usually remain in the mRNA transcribed from the expression unit of the vector and may reduce its stability or the efficiency of translation. Furthermore, one must be careful not to produce an ATG codon at the junctional sequence between the MCS and the insert, because it may work as a false start codon in the resulting mRNA and interfere with the production of the correct product. 

Immediately after the dATP chase add 1 pA restriction endonuclease (1 unit) to each sample in the capillaries. 

Restriction endonuclease (0.5-2 units) in appropriate amounts, and incubate under optimum conditions to obtain complete digestion (manufacturers recommendations) in a final volume of 20 /zl. 

BamHl (10 units//ul) with 10X reaction buffer supplied— We obtain this from Gibco BRL (catalog 15201-031), but any other supplier of quality restriction endonucleases will do. 

DNA molecules are too large to sequence as a unit, so DNA is first cleaved at specific base sequences and the resulting DNA fragments are sequenced. The enzymes that cleave DNA at specific base sequences are called restriction endonucleases, and the DNA fragments that are formed are called restriction fragments. Several hundred restriction enzymes are now known. A few examples of restriction enzymes, the base sequence each recognizes, and the point of cleavage in that base sequence are shown here. 

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