Restriction fragments, double-stranded

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

In your notebook, invent your own restriction enzyme. Show the DNA base sequence where it cleaves. Then construct a double stranded section of DNA with at least 25 base pairs and mark the points of cleavage by your enzyme. Finally, show all fragments formed. 

Type II site-specific deoxyribonuclease [EC 3.1.21.4], also referred to as type II restriction enzyme, catalyzes the endonucleolytic cleavage of DNA to give specific, double-stranded fragments with terminal 5 -phosphates. Magnesium ions are required as cofactors. 

Another method is to extract the DNA, then treat it with an enzyme that cleaves double-stranded DNA at specific sequences (a restriction enzyme see below). By partially digesting the DNA, large fragments can be generated moreover, these fragments will contain identical overlapping sequences at their ends that will help later in the cloning process. 

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. 

Restriction endonucleases catalyze the hydrolysis of specific phosphodi-ester bonds in double-stranded DNA. These enzymes are often used to linearize a circular plasmid for hybrid DNA construction. They have also found use in the analysis of DNA and the construction of restriction maps. In this experiment, students will incubate various restriction enzymes with plasmid or viral DNA and analyze the product DNA fragments by agarose gel electrophoresis. 

To incorporate fragments of foreign DNA into a plasmid vector, methods for cutting and rejoining of double-stranded DNA are required. Identification and manipulation of restriction endonucleases in the 1960s constituted a key discovery for cloning. Today, commonly used restriction enzymes are produced recombinantly and distributed widely. 

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