Saturday, 14 February 2015

RECOMBINANT DNA TECHNOLOGY: Definition, components & raw materials, enzymes used for the recombination, importance of vector and application.

Recombinant DNA technology is the core capability of bio nanotechnology. This technology allows us to construct any protein that we wish, simply by changing the genetic plans that are used to build it. Two natural enzymes— restriction enzymes and DNA ligase—are the keys to recombinant DNA technology, allowing us to edit the information in a DNA strand. Before the discovery of these enzymes, researchers modified the genetic code of living organisms by using biology’s own tools of mating and crossing or by random mutagenesis with chemicals or ionizing radiation. Today, researchers modify the genetic code rationally at the atomic level.

Restriction enzymes are unbelievably useful enzymes (I am reminded of the babble fish in Douglas Adams’s The Hitchhiker’s Guide to the Universe). They are built by bacteria to protect themselves from viral infection. The bacterium builds a restriction enzyme that cleaves DNA at one specific sequence. At the same time, it protects its own DNA by modifying the bases at this same sequence, so the restriction enzyme does not cleave its own genome. Invading viral DNA, however, is instantly chopped up by the restriction enzyme, because it is not similarly protected. Serendipitously, many restriction enzymes make staggered cuts in the two DNA strands, instead of cutting both strands straight across the DNA helix. Here is where biotechnology steps in with a new use for these enzymes. These ends are “sticky” and readily associate with other sticky ends of similar sequence. So restriction enzymes may be used to cut DNA, producing sticky ends that may be pasted back together in custom orientations. Thus restriction enzymes, originally evolved merely for their destructive capacity, are now tools for atomic-precision editing of large pieces of DNA.

Today, recombinant DNA technology has flowered. Clever researchers are continually discovering new methods for harnessing the protein production machinery of the cell in new ways. Consistent methods, often in the form of commercial kits, are available for every possible process. We can find and extract specific genes from organisms. We can duplicate and determine the sequence of large quantities of these genes. We can mutate, recombine, and splice these genes or create entirely new genes nucleotide by nucleotide. Finally, we can replace these genes into cells, modifying their genetic information.

DNA May Be Engineered with Commercially 

Available Enzymes

Customized DNA is routinely created in thousands of laboratories worldwide. Together, biological and synthetic techniques allow the construction of large DNA strands composed of natural DNA sequences or entirely new DNA sequences. A successful service industry has arisen that provides basic expertise for DNA manipulation. You can readily purchase stretches of DNA of any given sequence and all the enzymes needed to handle them. Researchers use a wide variety of natural bio-molecules for handling DNA. Well-characterized protocols and commercial sources for these enzymes are available, so these processes are available to any modest laboratory.
A few of the most important are:
(1) Restriction enzymes are isolated from bacteria. Over 100 types are available commercially. Each one cuts DNA at a specific sequence of bases. Typically, restriction enzymes are composed of two identical subunits, so they attack DNA symmetrically and cut at specific palindromic sequences.
(2) DNA ligase reconnects broken DNA strands. When two sticky ends anneal, DNA ligase is used to reconnect the breaks.
(3) DNA polymerase creates a new DNA strand by using another strand as a template, creating a double helix from a single strand. It is used to fill single-stranded gaps and to copy entire pieces of DNA.

Chemical synthesis of DNA perfectly complements these natural biomolecular tools for manipulating DNA. Current methods allow the automated synthesis of DNA strands about 100 nucleotides in length. Two complementary strands are easily constructed and annealed in solution to form a double helix. Short oligonucleotides are routinely synthesized and are available commercially. Once a new DNA is constructed, large quantities are produced by two major methods: DNA cloning and the polymerase chain reaction. The term “cloning” refers to the creation of identical copies without the normal processes of sexual reproduction: copies of mice or sheep, identical cultures of cells, or, in this case, many identical copies of a particular fragment of DNA. In DNA cloning, a bacterial cell is used to create many identical copies of a DNA sequence. One method is to insert the DNA sequence of interest into a virus, which then infects bacterial cells and forces them to make many copies. Alternatively, a bacterial plasmid may be used. Bacteria naturally contain small circles of DNA plasmids in addition to their main genome. To clone a DNA sequence, we add it to a bacterial plasmid and then insert it into bacteria. The plasmid is then copied each time the bacterium divides, forming large quantities of the DNA as the culture grows. The polymerase chain reaction (PCR) is a method for copying a small sample of DNA. It takes advantage of an efficient, heat-stable DNA polymerase isolated from bacteria that live in hot springs. PCR proceeds in cycles, doubling the number of DNA strands at each step.

PCR is so powerful you can start with a single strand of DNA and get as much as needed out. Once engineered DNA strands are built, we need methods to use them to create custom proteins. Proteins are conveniently made in engineered cells using expression vectors, plasmids that contain the gene specifying the protein along with a highly active promoter sequence. The promoter, which is often taken from a virus, directs the engineered cell to create large quantities of messenger RNA based on the plasmid DNA in the vector. The cell then synthesizes the protein based on this messenger RNA. Bacteria are the most widely utilized host cells that are engineered for protein production. Engineered bacteria create large amounts of protein, often comprising 1–10% of the total cellular protein. Also, bacteria are easy to grow, and inexpensive fermentation methods allow growth of high densities of bacterial cells with modest resources. However, bacteria present several significant limitations. Animal and plant cells often modify their proteins after they are synthesized, and bacteria do not perform these modifications. In particular, many animal and plant proteins have carbohydrate groups attached to their surfaces, and bacteria do not add these groups to engineered proteins. This can be a fatal problem in the production of proteins for use in medical treatment. Many of these proteins must have the appropriate carbohydrate groups to be active, and the immune system can react dangerously to improper carbohydrate groups (for instance, the need to be careful of blood types during transfusions is due to differences in the carbohydrates attached to cellular proteins). Engineered yeast cells, insect cells, or cultured mammalian cells may be used in cases where the proteins must be modified for proper action.

Another problem with engineered bacteria, which is occasionally an asset, is that the proteins tend to aggregate when they reach high concentrations, forming inclusion bodies. Inclusion bodies are dense aggregates of proteins that are easily visible in the microscope, often extending entirely across the bacterial cell. They are formed when new proteins associate randomly before they can undergo the proper folding process. Inclusion bodies are extremely tough, and harsh conditions must be used to solubilize the individual protein chains. In many cases, the purified proteins may then be folded under conditions that lead to the proper structure. If it is possible to renature the functional protein from inclusion bodies, they can be a substantial aid to purification. Because inclusion bodies are denser than most of the other structures in the cell, they are easily separated from the other cellular components simply by centrifuging the cell extract. Proteins may also be created without the help of living cells, by isolating the protein production machinery and performing the reactions in the test tube. The first step of protein production, the transcription of DNA into a messenger RNA, is now routine with purified RNA polymerase. However, the second step, the synthesis of proteins based on purified messenger RNA in cell-free systems, is still a technical challenge.

In some cases, extracts of the cell cytoplasm, containing the protein synthesis machinery along with everything else, are effective. Extracts can, however, encounter problems with limited energy supply and the presence of protease and nuclease enzymes that cleave the products and RNA message. Specialized continuous-flow cell-free systems have been developed to overcome this problem. Attempts to recreate protein synthesis with purified preparations of the components have also been successful. But because of the complexity of the system, requiring over 100 separate components, they are still limited to relatively modest yields. These methods are primarily used in research rather than industrial production of proteins. The advantages, however, of cell free protein production make it an attractive goal. It provides a controlled method for synthesizing proteins that are difficult in engineered bacteria, such as membrane-binding proteins, proteins that are toxic to bacteria, and proteins that include unusual amino acids. Development of efficient cell free translation mechanisms is an area of active research.

 Recombinant DNA technology relies on two key enzymes. Restriction enzymes, such as EcoRI shown on the left, cut DNA at specific sequences. Often, these enzymes make a staggered cut, producing “sticky ends,” as shown in the center. DNA ligase, shown on the right, connects two strands back together.

 The plasmid pBR322 is one of the most common vectors used to engineer the bacterium Escherichia coli. A map of the plasmid, which contains 4361 base pairs of DNA, is shown here. The plasmid contains a region that directs the replication of the plasmid (ori) and two genes that encode proteins for antibiotic resistance, one for ampicillin (ampR) and one for tetracycline (tetR). The sites that are cleaved by different restriction enzymes are shown surrounding the circle. By choosing the appropriate enzyme, the plasmid can be cut at specific locations. Researchers add new genes to the plasmid by cutting at one of the restriction sites and splicing in the new DNA. The drug-resistance genes provide a clever method of determining whether or not any bacteria have taken up the plasmid. For instance, if the new DNA is added at the PstI site at position 3607, the inserted DNA will disrupt the ampicillin-resistance gene. Thus bacteria that contain this new plasmid are easily identified and separated from bacteria that do not contain the plasmid: They will be resistant to tetracycline but sensitive to ampicillin

 Through repeated rounds of DNA synthesis and separation of the two strands, the polymerase chain reaction amplifies the amount of DNA in a sample. (1) The process begins with a single strand of DNA. (2) It is separated by heating, and short primer strands are added to the ends. (3) DNA polymerase builds a new strand using the separated strands as a template. (4) At the end of the cycle, there are two identical DNA double helices. This cycle is repeated, doubling the DNA at each step. The use of a heat-stable polymerase is the trick to making this an automated process, because it can survive the heating step of each cycle.


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