Recombinant DNA

Recombinant DNA
One of the most important tools in this technology is a series of enzymes called restriction endonucleases. Each of these enzymes, derived from bacteria, cleaves double-stranded DNA at particular sites determined by the particular base sequences at that point. Many of these endonucleases cut the DNA strands so that one has several bases projecting farther than the other strand (Figure 5-23), leaving what are called “sticky ends.” When these DNA fragments are mixed with others that have been cleaved by the same endonuclease, they tend to anneal (join) by the rules of complementary base pairing. They are sealed into their new position by the enzyme DNA ligase.

Besides their chromosomes, most prokaryote and at least some eukaryote cells have small circles of double-stranded DNA called plasmids. Though comprising only 1% to 3% of the bacterial genome, they may carry important genetic information, for example, resistance to an antibiotic. Plastids in plant cells (for example, chloroplasts) and mitochondria, found in most eukaryotic cells, are self-replicating and have their own complement of DNA in the form of small circles reminiscent of plasmids. The DNA of mitochondria and plastids codes for some of their proteins, and some of their proteins are specified by nuclear genes. If the DNA annealed after cleavage by the endonuclease is from two different sources, for example, a plasmid (see note above) and a mammal, the product is recombinant DNA. To make use of the recombinant DNA, the modified plasmid must be cloned in bacteria. The bacteria are treated with dilute calcium chloride to make them more susceptible to taking up the recombinant DNA, but the plasmids do not enter most of the cells present. Bacterial cells that have taken up the recombinant DNA can be identified if the plasmid has a marker, for example, resistance to an antibiotic. Then, the only bacteria that can grow in the presence of the antibiotic are those that have absorbed the recombinant DNA. Some bacteriophages (bacterial viruses) have also been used as carriers for recombinant DNA. Plasmids and bacteriophages that carry recombinant DNA are called vectors. The vectors retain the ability to replicate in the bacterial cells; therefore the recombinant insert is amplified.

A clone is a collection of individuals or cells all derived by asexual reproduction from a single individual.When we speak of cloning a gene or plasmid in bacteria,we mean that we isolate a colony or group of bacteria derived from a single ancestor into which the gene or plasmid was inserted.

Polymerase Chain Reaction Recent advances have made it a simple task to clone a specific gene enzymatically from any organism as long as part of the sequence of that gene is known. The technique is called the polymerase chain reaction (PCR). Two short chains of nucleotides called primers are synthesized; primers are complementary to different DNA strands in the known sequence. A large excess of each primer is added to a sample of DNA from the organism, and the mixture is heated to separate the double helix into single strands. When the mixture is cooled, there is a much greater probability that each strand of the gene of interest will anneal to a primer than to the other strand of the gene—because there is so much more primer present. DNA polymerase is added along with the four deoxyribonucleotide triphosphates, and DNA synthesis proceeds from the 3´ end of each primer, extending the primer in the 5´ to 3´ direction. If the primers are chosen so that each anneals toward the 3´ end of each of the complementary strands, entire new complementary strands will be synthesized, and the number of copies of the gene has doubled (Figure 5-24). The reaction mixture is then reheated and cooled again to allow more primers to bind original and new copies of each strand. With each cycle of DNA synthesis, the number of copies of the gene doubles. Since each cycle can take less than five minutes, the number of copies of a gene can increase from one to over one million in less than two hours! The PCR allows cloning a known gene from an individual patient, identification of a drop of dried blood at a crime scene, or cloning the DNA of a 40,000-year-old woolly mammoth.

Recombinant DNA technology and the PCR are currently being used in many areas with great positive potential and many practical uses.

The techniques of molecular biology have allowed scientists to accomplish feats of which few could dream only a decade or so ago.These accomplishments will bring enormous benefits for humanity in the form of enhanced food production and treatment of disease. Progress with crop plants has been so rapid that genetically engineered soybean, cotton, rice, corn, sugarbeet, tomato, and alfalfa have already reached the market in the United States. There is resistance to sale of genetically altered produce in Europe, apparently because of widespread fears that such vegetables can somehow harm consumers.

Development of transgenic animals of potential use has not progressed as far as development of such plants.Gene therapy for inherited diseases presents many difficulties, but research in this area is vigorous,and clinical trials for certain conditions are under way.