The Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture

If the nucleotide sequences at the ends of a particular DNA region are known, the intervening fragment can be amplified directly by the polymerase chain reaction (PCR). Here we describe the basic PCR technique and three situations in which it is used.

The PCR depends on the ability to alternately denature (melt) double-stranded DNA molecules and renature (anneal) complementary single strands in a controlled fashion. As in the membrane-hybridization assay described earlier, the presence of noncomplementary strands in a mixture has little effect on the base pairing of complementary single DNA strands or complementary regions of strands. The second requirement for PCR is the ability to synthesize oligonu-cleotides at least 18-20 nucleotides long with a defined sequence. Such synthetic nucleotides can be readily produced with automated instruments based on the standard reaction scheme shown in Figure 9-18.

As outlined in Figure 9-24, a typical PCR procedure begins by heat-denaturation of a DNA sample into single strands. Next, two synthetic oligonucleotides complementary to the 3' ends of the target DNA segment of interest are added in great excess to the denatured DNA, and the temperature is lowered to 50-60 °C. These specific oligonu-cleotides, which are at a very high concentration, will hybridize with their complementary sequences in the DNA sample, whereas the long strands of the sample DNA remain apart because of their low concentration. The hybridized oligonucleotides then serve as primers for DNA chain synthesis in the presence of deoxynucleotides (dNTPs) and a temperature-resistant DNA polymerase such as that from Thermus aquaticus (a bacterium that lives in hot springs). This enzyme, called Taq polymerase, can remain active even after being heated to 95 °C and can extend the primers at temperatures up to 72 °C. When synthesis is complete, the whole mixture is then heated to 95 °C to melt the newly formed DNA duplexes. After the temperature is lowered again, another cycle of synthesis takes place because excess primer is still present. Repeated cycles of melting (heating) and synthesis (cooling) quickly amplify the sequence of interest. At each cycle, the number of copies of the sequence between the primer sites is doubled; therefore, the desired sequence increases exponentially—about a million-fold after 20 cycles—whereas all other sequences in the original DNA sample remain unamplified.

Direct Isolation of a Specific Segment of Genomic DNA

For organisms in which all or most of the genome has been sequenced, PCR amplification starting with the total ge-nomic DNA often is the easiest way to obtain a specific DNA region of interest for cloning. In this application, the two oligonucleotide primers are designed to hybridize to sequences flanking the genomic region of interest and to include sequences that are recognized by specific restriction enzymes (Figure 9-25). After amplification of the desired target sequence for about 20 PCR cycles, cleavage with the appropriate restriction enzymes produces sticky ends that allow efficient ligation of the fragment into a plasmid vector cleaved by the same restriction enzymes in the polylinker. The resulting recombinant plasmids, all carrying the identical genomic DNA segment, can then be cloned in

Region to be amplified

Primer 1

DNA synthesis

Round 1 S'

Round 2

DNA synthesis

Round 3

HindIW site

Continue for -20 PCR cycles

Cut with restriction enzymes

Sticky end

Sticky end

Ligate with plasmid vector with sticky ends

▲ EXPERIMENTAL FIGURE 9-25 A specific target region in total genomic DNA can be amplified by PCR for use in cloning. Each primer for PCR is complementary to one end of the target sequence and includes the recognition sequence for a restriction enzyme that does not have a site within the target region. In this example, primer 1 contains a BamHI sequence, whereas primer 2 contains a HindIII sequence. (Note that for clarity, in any round, amplification of only one of the two strands is shown, the one in brackets.) After amplification, the target segments are treated with appropriate restriction enzymes, generating fragments with sticky ends. These can be incorporated into complementary plasmid vectors and cloned in E. coli by the usual procedure (see Figure 9-13).

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