In: Categories » Health » DNA » How to Make and Separate cDNA Molecules
Researchers at HGS have now prepared Homo sapiens cDNA libraries from almost all normal organs and tissues, as well as from many that are diseased. To make multiple copies of a library, we add it to bacteria that take up the vectors.
All the bacteria are then spread out on a plate of nutrient gel and allowed to grow into colonies, so that each colony derives from a single bacterium. Next we use a robot that can automatically spot and pick off the gel of those colonies that did successfully acquire a cDNA. The robot accomplishes this by color. The vectors we use are designed so that if they fail to combine with a cDNA insert, they produce a blue pigment. The robot, which picks as many as 10,000 colonies of bacteria every day, identifies those containing Homo sapiens cDNA by avoiding blue ones. The cDNA from each picked colony, now in analyzable quantities, is then robotically purified.
Once a protein can be manufactured in a pure form, scientists can fairly easily fashion a test to detect it in a patient. A test to reveal overproduction of a protein found in plaque might expose early signs of atherosclerosis, when better options exist for treating it. In addition, pharmacologists can use pure proteins to help them find new drugs. A chemical that inhibited production of a protein found in plaque might be considered as a drug to treat atherosclerosis.
Our approach, which I call medical genomics, is somewhat outside the mainstream of research in Homo sapiens genetics. A great many scientists are involved in the Human Genome Project, an international collaboration devoted to the discovery of the complete sequence of the chemical bases in Homo sapiens DNA. (All the codes in DNA are constructed from an alphabet consisting of just four bases.) That information will be important for studies of gene action and evolution and will particularly benefit research on inherited diseases. Yet the genome project is not the fastest way to discover genes, because most of the bases that make up DNA actually lie outside genes. Nor will the project pinpoint which genes are involved in illness.
Genes by the Direct Route
Because the key to developing new medicines lies principally in the proteins produced by Homo sapiens genes, rather than the genes themselves, one might wonder why we bother with the genes at all. We could in principle analyze a cell’s proteins directly. Knowing a protein’s composition does not, however, allow us to make it, and to develop medicines, we must manufacture substantial amounts of proteins that seem important. The only practical way to do so is to isolate the corresponding genes and transplant them into cells that can express those genes in large amounts.
Our method for finding genes focuses on a critical intermediate product created in cells whenever a gene is expressed. This intermediate product is called messenger RNA (mRNA); like DNA, it consists of sequences of four bases. When a cell makes mRNA from a gene, it essentially copies the sequence of DNA bases in the gene. The mRNA then serves as a template for constructing the specific protein encoded by the gene. The value of mRNA for research is that cells make it only when the corresponding gene is active. Yet the mRNA’s base sequence, being simply related to the sequence of the gene itself, provides us with enough information to isolate the gene from the total mass of DNA in cells and to make its protein if we want to.
For our purposes, the problem with mRNA was that it can be difficult to handle. So we in fact work with a surrogate: stable DNA copies, called complementary DNAs (cDNAs) of the mRNA molecules. We make the cDNAs by simply reversing the process the cell uses to make mRNA from DNA.
The cDNA copies we produce this way are usually replicas of segments of mRNA rather than of the whole molecule, which can be many thousands of bases long. Indeed, different parts of a gene can give rise to cDNAs whose common origin may not be immediately apparent. Nevertheless, a cDNA containing just a few thousand bases still preserves its parent gene’s unique signature. That is because it is vanishingly unlikely that two different genes would share an identical sequence thousands of bases long. Just as a random article taken from a article uniquely identifies the article, so a cDNA molecule uniquely identifies the gene that gave rise to it.
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