hybridization has many uses in modern cell and molecular biology; one of the most powerful is in the cloning of DNA by the polymerase chain reaction, as we next discuss.
Genomic and cDNA libraries were once the only route to cloning genes and they are still used for cloning very large genes and for sequencing whole genomes. However, a powerful and versatile method for amplifying DNA, known as the polymerase chain reaction (PCR), provides a more rapid and straightforward approach to DNA cloning, particularly in organisms whose complete genome sequence is known. Today, since genome sequences are abundant, most cloning is carried out by PCR.
Invented in the 1980s, PCR revolutionized the way that DNA and RNA are analyzed. The technique can amplify any nucleotide sequence selectively and is performed entirely in a test tube. Eliminating the need for bacteria makes PCR convenient and rapid—billions of copies of a nucleotide can be generated in a matter of hours. Starting with an entire genome, PCR allows DNA from a specified region—selected by the experimenter—to be greatly amplified, effectively “purifying” this DNA away from the remainder of the genome, which remains unamplified. Because of its power to greatly amplify nucleic acids, PCR is remarkably sensitive: the method can be used to detect the trace amounts of DNA in a drop of blood left at a crime scene or in a few copies of a viral genome in a patient’s blood sample.
The success of PCR depends both on the selectivity of DNA hybridization and on the ability of DNA polymerase to copy a DNA template faithfully through repeated rounds of replication in vitro. As discussed in Chapter 5, this enzyme adds nucleotides to the 3ʹ end of a growing strand of DNA (see Figure 5–4). To copy DNA, the polymerase requires a primer—a short nucleotide sequence that provides a 3ʹ end from which synthesis can begin. For PCR, the primers are designed by the experimenter, synthesized chemically, and, by hybridizing to genomic DNA, “tell” the polymerase which part of the genome to copy. As discussed in the previous section, DNA primers (in essence, the same type of molecules as DNA probes but without a radioactive or fluorescent label) can be designed to uniquely locate any position on a genome.
PCR is an iterative process in which the cycle of amplification is repeated dozens of times. At the start of each cycle, the two strands of the double-stranded DNA template are separated and a different primer is annealed to each. These primers mark the right and left boundaries of the DNA to be amplified. DNA polymerase is then allowed to replicate each strand independently (Figure 8–35). In
Figure 8–35 A pair of primers directs the synthesis of a desired segment of DNA in a test tube. Each cycle of PCR includes three steps: (1) The double-stranded DNA is heated briefly to separate the two strands. (2) The DNA is exposed to a large excess of a pair of specific primers— designed to bracket the region of DNA to be amplified—and the sample is cooled to allow the primers to hybridize to complementary sequences in the two DNA strands. (3) This mixture is incubated with DNA polymerase and the four deoxyribonucleoside triphosphates so that DNA can be synthesized, starting from the two primers. To amplify the DNA, the cycle is repeated many times by reheating the sample to separate the newly synthesized DNA strands (see Figure 8–36).
The technique depends on the use of a special DNA polymerase isolated from a thermophilic bacterium; this polymerase is stable at much higher temperatures than eukaryotic DNA polymerases, so it is not denatured by the heat treatment shown in step 1. The enzyme therefore does not have to be added again after each cycle.
products of frst cycle