Blog Against Cancer

RESTRICTION ENDONUCLEASESAND RECOMBINANT DNA

In eukaryotic chromosomes, individual molecules of DNA are several million base pairs long. Because these molecules are far too large to analyze directly, scientists are usually interested in cutting DNA into fragments of manageable size. Fortunately, for molecular biologists, bacteria have evolved a highly diverse set of enzymes, the “restriction endonucleases,” that cleave DNA internally within the polymer. In nature, these enzymes have evolved to protect the bacteria from invasion by foreign DNA molecules, such as phage. In order to discriminate between “domestic” and “foreign” DNA, these enzymes recognize specific nucleotide sequences. DNA without such specific sequences is left undisturbed by the enzymes. However, when a
restriction endonuclease spots a “recognition site,” it binds to the site and cleaves both strands of the DNA to which it has bound.

Individual restriction endonucleases recognize specific sequences, usually in the order of four to six bases in length, and these sequences are often palindromes, that is, the 5 ′ to 3′ sequence in the upper strand is identical to the 5 ′ to 3′ sequence in the lower strand . While restriction endonucleases cut DNA into smaller fragments,
there is a lower limit to the size of useful fragments. One would not want to cut DNA into such small pieces that the informational content of each piece is negligible. Statistically, the longer a restriction endonuclease’s recognition sequence, the less frequently this sequence will occur in a stretch of DNA. Therefore, the enzymes most commonly used to cut DNA into usefully large fragments are those that recognize a six-nucleotide recognition site (so-called “six-base cutters”). For example, an endonuclease isolated from Escherichia coli, called EcoRI, recognizes the sequence GAATTC, and wherever this occurs in double-stranded DNA, it will cleave between the G and A (see Figure ). (Note that the antisense strand, which reads CTTAAG in the 3′ to 5 ′ direction, will also read GAATTC in the 5′ to 3′ direction.
This is what is meant by a palindromic sequence).

GENE CLONING Mechanics.

The most powerful technique available for gene analysis, and the one technique that is the cornerstone for
all others, is gene cloning . In the gene cloning process, a discrete piece of DNA is faithfully replicated in the laboratory. Cloning provides quantities of specific DNA sufficient for biochemical analysis or for any other manipulation, including joining to a foreign piece of DNA. In the early 1970s, Cohen and Boyer drew upon
two fundamental properties of bacteria and their viruses (phages) that made this innovation possible: plasmids and DNA ligases.Plasmids are circular molecules of DNA that replicate in the cytoplasm of bacterial cells, separate from the bacteria’s own DNA. In nature, plasmids often carry genetic information useful to the host bacterium, such as genes that confer resistance to antibiotics. For the purposes of gene cloning, plasmids are important because they contain all the information necessary for directing bacterial enzymes to replicate the plasmid DNA, in some cases, to many thousands of copies per bacterium.

DNA ligases are enzymes produced by bacteria (and some phages when they infect bacteria) that can link or ligate together separate pieces of DNA. The nucleotide sequence in a piece of DNA does not influence the activity of a DNA ligase so that a DNA ligase can join two pieces of DNA that are not ordinarily connected to each other in nature.
In gene cloning, one uses a restriction endonuclease to cut open the circular plasmid DNA in a region of the plasmid not necessary for replication. Suppose, for example, that the enzyme EcoRI cuts open the plasmid in such a nonessential area. EcoRI recognizes the sequence GAATTC, and cuts both DNA strands between the G and the A nucleotides. Protruding from the cut ends will be single- stranded DNA “tails” having the sequences AATT. (Note that the tail’s sequence in the sense strand is the same as the sequence in the antisense strand when the nucleotides are read in the 5′ to 3′ direction). Any other piece of DNA that has been cut with EcoRI will also have single-stranded AATT tails, and the AATT tails on this foreign piece of DNA can base-pair with the complementary TTAA tails (reading 3′ to 5 ′) on the cut plasmid. When this happens, the foreign DNA piece physically closes the gap in the plasmid, forming a closed circular plasmid again (which is necessary for plasmid propagation).

Although the nucleotides at the ends of the plasmid and foreign DNA now abut each other, they are not covalently connected. This is an unstable situation which the DNA ligase rectifies. The DNA ligase covalently joins the plasmid and foreign DNA to create a “recombinant” plasmid which still has all the information needed to be replicated in a bacterium, but which also contains a foreign DNA “insert.” Obviously, the EcoRI-cut ends of the plasmid can also base-pair with themselves again to re-form the native plasmid, but molecular biologists have developed a number of tricks to suppress this phenomenon. It should be pointed out that single-stranded tails are not always necessary for making recombinant DNA. Under certain conditions, the DNA ligase can join together two fragments of “blunt-ended” DNA without these tails.

When a recombinant plasmid is re-introduced into a host bacterium (by a process called “transformation”), the plasmid will replicate normally. Now, however, its foreign DNA insert is replicated along with the plasmid into which it was inserted. The transformed bacteria can then be grown to large numbers in liquid culture. With each bacterial cell division, the progeny bacteria contain plasmid molecules that continue to replicate. When the bacterial culture contains the desired quantity of this plasmid (this may be milligrams of
plasmid DNA in a one-liter culture), it can be reisolated as pure DNA.

The cloned foreign piece of DNA can then be cut out  for further analysis or manipulation. One can also use
bacterial viruses (or phages) in the same manner by infecting host bacteria with recombinant phage bearing foreign DNA sequences. In all these experiments, the plasmid or phage that houses the foreign DNA
is called a “vector,” because it is the vehicle that directs the foreign DNA into the host bacterium.
These extraordinarily powerful tools, which are now part of the standard armamentarium of all molecular biology laboratories, have been responsible for the development of nearly all the analytical techniques
described below. Several excellent manuals have been published that describe these techniques in detail.