Working at the Molecular Level

The manipulation of genes presents a serious challenge, often requiring strategies that may not, at first, seem obvi ous. The basic problem is that genes are minute and there are thousands of them in every cell. Even when viewed with the most powerful microscope, DNA appears as a tiny thread—individual nucleotides cannot be seen, and no physical features mark the beginning or the end of a gene.

To illustrate the problem, let's consider a typical situation faced by a molecular geneticist. Suppose we wanted to isolate a particular human gene, place it inside bacterial cells, and use the bacteria to produce large quantities of the encoded human protein. The first and most formidable problem is to find the desired gene. A haploid human genome consists of 3.3 billion base pairs of DNA. Let's assume that the gene that we want to isolate is 3000 bp long. Our target gene occupies only one-millionth of the genome; so searching for our gene in the huge expanse of genomic DNA is more difficult than looking for a needle in the proverbial haystack. But, even if we are able to locate the gene, how are we to separate it from the rest of the DNA? No forceps are small enough to pick up a single piece of DNA, and no mechanical scissors precise enough to snip out an individual gene.

If we did succeed in locating and isolating the desired gene, we would next need to insert it into a bacterial cell. Linear fragments of DNA are quickly degraded by bacteria; so the gene must be inserted in a stable form. It must also be able to successfully replicate or it will not be passed on when the cell divides.

If we succeed in transferring our gene to bacteria in a stable form, we still must ensure that the gene is properly transcribed and translated. Gene expression is a complex process requiring a number of DNA sequence elements, some of which lie outside the gene itself (Chapters 13 through 16). All of these elements must be present in their proper orientations and positions for the protein to be produced.

Finally, the methods used to isolate and transfer genes are inefficient and, of a million cells that are subjected to these procedures, only one cell might successfully take up and express the human gene. So we must search through many bacterial cells to find the one containing the recombinant DNA. We are back to the problem of the needle in the haystack.

Although these problems might seem insurmountable, molecular techniques have been developed to overcome all of them, and human genes are routinely transferred to bacterial cells, where the genes are expressed. __

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