Restriction enzymes, also known as restriction endonucleases, are enzymes that cut a
at a particular place. They are essential tools for
. The enzyme “scans” a DNA molecule, looking for a particular sequence, usually of four to six nucleotides. Once it finds this recognition sequence, it stops and cuts the strands. This is known as enzyme digestion. On double stranded DNA the recognition sequence is on both strands, but runs in opposite directions. This allows the enzyme to cut both strands. Sometimes the cut is blunt, sometimes the cut is uneven with dangling nucleotides on one of the two strands. This uneven cut is known as sticky ends.
A blunt end may look like this:
A sticky end like this:
Most plasmids used for recombinant technology have recognition sequences for a number of restriction enzymes. This allows a scientist to choose from a number of places to cut the plasmid with a restriction enzyme. Ligation enzymes can then be used to sort of paste in new genomic sequences. These mutated, or recombined, plasmids can then be grown up in bacterial cells and used for a number of purposes, including the addition of genes to mammalian genomes.
You always want to read carefully the information sheet that comes with your enzymes as well as the catalogue information. The better you know your enzyme, the more likely you will be to have a successful digestion. Most enzymes come in glycerol solution as a storage buffer, but enzymes don’t work well in the presence of high glycerol concentration. You want to be sure to dilute the glycerol content down to less than 5% to ensure proper enzymatic activity.
Problems with enzyme activity can occur under the following conditions:
Some other helpful tips for working with enzymes include:
Although scientists as far back in history as Aristotle recognized that the features of one generation are passed on to the next (…like begets like…) it was not until the 1860′s that the fundamental principles of genetic inheritance were described by Gregor Mendel. Mendel’s work with common garden peas, pisum sativum, led him to hypothesize that phenotypic traits (physical characteristics) are the result of the interaction of discrete particles, which we now call genes, and that both parents provide particles which make up the characteristics of the offspring. His theories were, however, widely disregarded by scientists of the time. In the last quarter of the 19th century, however, microscopists and cytologists, interested in the process of cell division, developed both the equipment and the methods needed to visualize chromosomes and their division in the processes of mitosis (A. Schneider, 1873) and of meiosis (E. Beneden, 1883).
As the 20th century began many scientists noticed similarities in the theoretical behavior of Mendel’s particles, and the visible behavior of the newly discovered chromosomes. It wasn’t long before most scientists were convinced that the hereditary material responsible for giving living things their characteristic traits, and chromosomes must be one in the same. Yet, questions still remained. Chemical analysis of chromosomes showed them to be composed of both protein and DNA. Which substance carried the hereditary information? For many years most scientists favored the hypothesis that protein was the responsible molecule because of its comparative complexity when compared with DNA. After all, DNA is composed of a mere 4 subunits while protein is composed of 20, and DNA molecules are linear while proteins range from linear to multiply branched to globular. It appeared clear that the relatively simple structure of a DNA molecule could not carry all of the genetic information needed to account for the richly varied life in the world around us!
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DNA Double Helix: A Recent Discovery of Enormous Complexity
The DNA Double Helix is one of the greatest scientific discoveries of all time. First described by James Watson and Francis Crick in 1953, DNA is the famous molecule of genetics that establishes each organism’s physical characteristics. It wasn’t until mid-2001, that the Human Genome Project and Celera Genomics jointly presented the true nature and complexity of the digital code inherent in DNA. We now understand that each human DNA molecule is comprised of chemical bases arranged in approximately 3 billion precise sequences. Even the DNA molecule for the single-celled bacterium, E. coli, contains enough information to fill all the books in any of the world’s largest libraries.
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