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Tandem Repeats and Morphological Variation. Chemical Structure of RNA. Eukaryotic Genome Complexity. RNA Functions. Restriction Enzymes By: Leslie A. Pray, Ph. Citation: Pray, L. Nature Education 1 1 Restriction enzymes are one of the most important tools in the recombinant DNA technology toolbox. But how were these enzymes discovered?
And what makes them so useful? Aa Aa Aa. When I come to the laboratory of my father, I usually see some plates lying on the tables. These plates contain colonies of bacteria. To be able to clone a DNA insert into a cloning or expression vector, both have to be treated with two restriction enzymes that create compatible ends. At least one of the enzymes used should be a sticky end cutter to ensure that the insert is incorporated in the right orientation.
It will save you a lot of time when you could carry out the two digestions simultaneously double digestion. Not all restriction enzymes work equally well in all commercially available buffers and, therefore, it is worthwhile to check e.
To ensure efficient digestion the two recognition sites should be more than 10 base pairs apart. If one of the enzymes is a poor cutter or if the sites are separated 10 base pairs or less, the digestions should be performed sequentially. The first digest should be done with the enzyme that is the poorest cutter and the second enzyme added after digestion has been verified by running a sample of the reaction mix on an agarose gel.
Scientists have identified and purified hundreds of different types of restriction enzymes. They are named after the genus and species of the organism they were isolated from and are given a number to indicate the order in which they were found. DNA consists of two complementary strands of nucleotides that spiral around each other in a double helix. Sma I is an example of a restriction enzyme that cuts straight through the DNA strands, creating DNA fragments with a flat or blunt end.
Other restriction enzymes, like Eco RI , cut through the DNA strands at nucleotides that are not exactly opposite each other. This creates DNA fragments with one nucleotide strand that overhangs at the end. This overhanging nucleotide strand is called a sticky end because it can easily bond with complementary DNA fragments.
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