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...........Recombinant DNA............
In the early 1970s, biochemists at Stanford University showed that genetic traits could indeed be transferred from one organism to another. In this experiment, the DNA of one microorganism recombined with the inserted DNA sequence of another, and thus had been edited to exhibit a very specific modification.......
To understand the significance and the various applications of biotechnology, a basic knowledge of rDNA technology is indispensable. The following description of cell processes may seem bewildering, but rDNA technology is made possible only through natural biological activities that are breathtaking in their elegance and efficiency.
One popular approach to discussing DNA is to use the analogy of language. Viewed as the foundation of a communication system, DNA may be thought of as the repository of all information needed to make an organism. (In some viruses, this information is stored in the ribonucleic acid, or RNA. For purposes of this discussion, however, such organisms will not be considered.)
The structure of the DNA molecule itself is identical among all living things, from an amoeba to a 150-ton whale, from a blade of grass to a redwood tree. It consists of sugar, phosphate, and four nitrogen bases: adenine (A), thymine (T), guanine (G), and cytosine (C). A sugar, phosphate, and base together constitute a nucleotide. The four bases are paired on the DNA molecule, and in a very specific way: A always with T and G always with C. Connecting the base pairs are alternating sugar and phosphate units, forming a structure that resembles a ladder. The ladder is actually three-dimensional, though; it takes the form of two strands twisted into a long spiral - the famous "double helix."
How is it that this molecule consisting of only six basic components (four bases, a phosphate, and a sugar) can contain all the information required to make almost a million types of animals and nearly a half-million species of plants?
Let us think of DNA as the totality of information needed to reproduce any organism. It is, in a manner of speaking, a language. Now consider: If we compiled all the information available to us - everything that has been discovered and created since man began to wonder about himself and the world around him - we would have an unimaginably vast body of data. But this superabundance of information would be useless until we imposed some order on it, much as the Dewey decimal system categorizes the books in a library, or the rules of grammar render language intelligible. By a similar process, all the random information in the DNA molecule is made specific and meaningful through the very precise ordering of the A, T, G, and C bases.
Since these four bases can pair up in only one way, their ordering on one strand of the DNA ladder dictates their ordering on the second strand. In the process of reproduction, the two strands unwind, and each then serves as the template (or foundation) for generating a new strand; the only possible result (barring some chance genetic mistake) is two DNA molecules that are identical in base pairings to the original molecule.
The bases individually convey no message. Instead, they act in strings of three, called codons. With a little calculation, we can figure out that four bases can be arranged in only sixty-four unique sets of three. But DNA's store of information comprises more than just four bases arranged into sixty-four different codons; just as the words of a language can be put together to form an infinite number of texts, so the codons on the DNA molecule can be ordered in innumerable ways.
What is the function of the codons on the DNA molecule? To give instructions for specifying and ordering amino acids. Amino acids are the structural elements of proteins, which in turn are the basic biochemical units that drive all biological processes. There are only twenty amino acids found in proteins, and the codes for ordering them are universal - the sequence of the nucleotide letters to specify the amino acid words is the same for elephants, lilac trees, and two-toed sloths. But the amino acid words can be combined in many ways to make thousands of protein sentences with distinct functions. Certain proteins, called enzymes, are catalysts (agents that are necessary for a reaction to occur but are not themselves changed in the process); others, called structural proteins, help to build cells and tissues.
If DNA can be thought of as the language of life, then the four bases can be seen as letters and the codons as arrangements of letters, or words. But like English, DNA's language is more than words. Some codons function as punctuation marks, containing instructions to stop or start manufacturing a protein. This chemically simple yet stunningly complex DNA molecule dictates not only what proteins the organism will be made of, but how these proteins are to be arranged.
We have seen that one codon contains the instructions for one amino acid, and that sequences of codons specify the production of proteins. Groups of codons that have been arranged in "grammatically" correct sentences to form specific proteins are called "genes".
All right, so DNA contains all the information needed to perpetuate life. This information builds in complexity from nucleotides to codons to genes, ultimately giving the complete text - the specific information to make a specific organism. But how do we get from having information to using it? It is the genes, the blueprints for making proteins, that finally get down to the business of taking all this information and doing something with it.
Through a complicated series of biochemical processes, the instructions contained in the genes are translated into the actual stuff of which organisms are made. It is at this point, when meaning becomes reality, that rDNA technology enters the picture.
Going back to our favorite analogy of DNA as a language, with letters, words, sentences, and punctuation marks forming a coherent text, one can ask whether it would be possible to edit the genetic text, modifying it to produce a desired result. In the early 1970s, biochemists at Stanford University showed that genetic traits could indeed be transferred from one organism to another. In this experiment, the DNA of one microorganism recombined with the inserted DNA sequence of another, and thus had been edited to exhibit a very specific modification.
The actual editing, or insertion process, is painstaking, for it involves manipulating incredibly tiny pieces of incredibly tiny organisms. But the process can be explained in terms of editing a written text: scissors and "glue" are used to "cut" and "paste."
The methods used in rDNA technology are fairly simple. We take, for example, the sentence (gene) for insulin production in humans and paste it into the DNA of Escherichia coli, a bacterium that inhabits the human digestive tract. The bacterial cells divide very rapidly making billions of copies of themselves, and each bacterium carries in its DNA a faithful replica of the gene for insulin production. Each new E. coli cell has inherited the human insulin gene sentence.
How do we transfer the gene embodying the instruction for insulin production? One approach would be to cut the appropriate gene from human DNA and paste, or splice, it into plasmid DNA, a special kind of DNA that takes a circular form and can be used as a vehicle for this editing job. Our "scissors" are the class of enzymes called restriction enzymes. There are well over a hundred restriction enzymes, each cutting in a very precise way a specific base sequence of the DNA molecule. With these scissors used singly or in various combinations, the segment of the human DNA molecule that specifies insulin production can be isolated. This segment is "glued" into place using an enzyme called DNA ligase. The result is an edited, or recombinant, DNA molecule. When this recombinant plasmid DNA is inserted into E. coli, the cell will be able to process the instructions to assemble the amino acids for insulin production. More importantly, the new instructions are passed along to the next generation of E. coli cells in the process known as gene cloning.
This module will familiarize the student with the basic tools and concepts used by molecular biologists in their scientific work. The biological workhorse in these experiments is the plasmid, a small piece of engineered DNA which contains a genetic element (gene) that can be expressed by the bacterial host into which it is inserted. The concepts and techniques taught in this unit are:
Basic genetic engineering (GE) takes donor DNA from one organism or type of cell and places it into the DNA of another organism or type of cell..
recombinant dna steps:
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6. Target Cells ReproduceMany recombined plasmids are inserted into many bacteria cells. While they live, the bacteria's cell processes turn on the inserted gene and the protein is produced in the cell. When the bacterial cells reproduce by dividing, the inserted gene is also reproduced in the newly created cells. |
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How do we transfer the gene embodying the instruction for insulin production? One approach would be to cut the appropriate gene from human DNA and paste, or splice, it into plasmid DNA, a special kind of DNA that takes a circular form and can be used as a vehicle for this editing job. Our "scissors" are the class of enzymes called restriction enzymes. There are well over a hundred restriction enzymes, each cutting in a very precise way a specific base sequence of the DNA molecule. With these scissors used singly or in various combinations, the segment of the human DNA molecule that specifies insulin production can be isolated. This segment is "glued" into place using an enzyme called DNA ligase. The result is an edited, or recombinant, DNA molecule. When this recombinant plasmid DNA is inserted into E. coli, the cell will be able to process the instructions to assemble the amino acids for insulin production. More importantly, the new instructions are passed along to the next generation of E. coli cells in the process known as gene cloning.
To be useful, the recombinant molecule must be replicated many times to provide material for analysis, sequencing, etc. Producing many identical copies of the same recombinant molecule is called cloning. Cloning can be done in vitro, by a process called the polymerase chain reaction (PCR). Here, however, we shall examine how cloning is done in vivo.
Cloning in vivo can be done inIn every case, the recombinant DNA must be taken up by the cell in a form in which it can be replicated and expressed. This is achieved by incorporating the DNA in a vector. A number of viruses (both bacterial and of mammalian cells) can serve as vectors. But here let us examine an example of cloning using E. coli as the host and a plasmid as the vector.
Using procedures like this, many human genes have been cloned in E. coli or in yeast. This has made it possible — for the first time — to produce unlimited amounts of human proteins in vitro. Cultured cells (E. coli, yeast, mammalian cells) transformed with the human gene are being used to manufacture: