Working with Gene Clones
Once a gene has been successfully cloned, a variety of procedures are available to characterize it.
Getting Enough DNA to Work with: The Polymerase Chain Reaction
Once a particular gene is identified within the library of DNA fragments, the final requirement is to make multiple copies of it. One way to do this is to insert the identified fragment into a bacterium; after repeated cell divisions, millions of cells will contain copies of the fragment. A far more direct approach, however, is to use DNA polymerase to copy the gene sequence of interest through the polymerase chain reaction (PCR; figure 8). Kary Mullis developed PCR in 1983 while he was a staff chemist at the Cetus Corporation; in 1993, it won him the Nobel Prize for chemistry. PCR can amplify specific sequences or add sequences (such as endonuclease recognition sequences) as primers to cloned DNA. There are three steps in PCR:
Step 1: Denaturation.First, an excess of primer (typically a synthetic sequence of 20 to 30 nucleotides) is mixed with the DNA fragment to be amplified. This mixture of primer and fragment is heated to about 98° C. At this temperature, the double-stranded DNA fragment dissociates into single strands.
Step 2: Annealing of Primers.Next, the solution is allowed to cool to about 60° C. As it cools, the single strands of DNA reassociate into double strands. However, because of the large excess of primer, each strand of the fragment base-pairs with a complementary primer flanking the region to be amplified, leaving the rest of the fragment single-stranded.
Step 3: Primer Extension.Now a very heat-stable type of DNA polymerase, called Taq polymerase (after the thermophilic bacterium Thermus aquaticus, from which Taq is extracted) is added, along with a supply of all four nucleotides. Using the primer, the polymerase copies the rest of the fragment as if it were replicating DNA. When it is done, the primer has been lengthened into a complementary copy of the entire single-stranded fragment. Because both DNA strands are replicated, there are now two copies of the original fragment.
Steps 13 are now repeated, and the two copies become four. It is not necessary to add any more polymerase, as the heating step does not harm this particular enzyme. Each heating and cooling cycle, which can be as short as 1 or 2 minutes, doubles the number of DNA molecules. After 20 cycles, a single fragment produces more than one million (220) copies! In a few hours, 100 billion copies of the fragment can be manufactured.
PCR, now fully automated, has revolutionized many aspects of science and medicine because it allows the investigation of minute samples of DNA. In criminal Figure 8
The polymerase chain reaction. (1) Denaturation. A solution containing primers and the DNA fragment to be amplified is heated so that the DNA dissociates into single strands. (2) Annealing of primers. The solution is cooled, and the primers bind to complementary sequences on the DNA flanking the region to be amplified. (3) Primer extension. DNA polymerase then copies the remainder of each strand, beginning at the primer. Steps 13 are then repeated with the replicated strands. This process is repeated many times, each time doubling the number of copies, until enough copies of the DNA fragment exist for analysis.
investigations, “DNA fingerprints” are prepared from the cells in a tiny speck of dried blood or at the base of a single human hair. Physicians can detect genetic defects in very early embryos by collecting a few sloughed-off cells and amplifying their DNA. PCR could also be used to examine the DNA of historical figures such as Abraham Lincoln and of now-extinct species, as long as even a minuscule amount of their DNA remains intact.