Molecular Biotechnology. Bernard R. Glick
DNA and then repeated the process with fresh polymerase, hypothesizing that after each cycle the number of molecules carrying the specific sequence between the primers would double. Despite the skepticism of his colleague, Mullis proved that his reasoning was correct, albeit the hard way. By manually cycling the reaction through temperatures required to denature the DNA and anneal and extend the oligonucleotides, each time adding a fresh aliquot of a DNA polymerase isolated from E. coli, he was able to synthesize unprecedented amounts of target DNA (Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263–273, 1986). Thermostable DNA polymerases that obviate the need to add fresh polymerase after each denaturation step and automated cycling have since made PCR a routine and indispensable laboratory procedure.
The capability of generating large amounts of DNA by amplification from segments of cloned or genomic DNA has facilitated the cloning of DNA versions of rare mRNA molecules, screening gene libraries, diagnostic testing for gene mutations, sequencing of genomes, and a myriad of other applications. In fact, the first study using PCR described a diagnostic test for sickle-cell anemia (Saiki et al., Science. 230:1350–1354, 1985). Mullis received the Nobel Prize in Chemistry for his work on PCR in 1993.
Amplification of DNA Using PCR
The essential components for PCR amplification are (i) a template sequence in a DNA sample that is targeted for amplification and is from 100 to 3,000 bp in length (larger regions can also be amplified, but with reduced efficiency); (ii) two synthetic oligonucleotide primers (∼20 nucleotides each) that are complementary to regions on opposite strands that flank the target DNA sequence and that, after annealing to the sample DNA, have their 3′ hydroxyl ends oriented toward each other; (iii) a thermostable DNA polymerase that remains active after repeated heating to 95°C or higher and copies the DNA template with high fidelity; (iv) the four deoxyribonucleotides; and (v) a reaction buffer that provides optimal pH and osmotic conditions, and cofactors (e.g., magnesium) required for DNA polymerase activity.
Replication of a specific DNA sequence by PCR requires three successive steps as outlined below. Amplification is achieved by repeating the three-step cycle 25 to 40 times. All steps in a PCR cycle are carried out in an automated block heater that is programmed to change temperatures after a specified period of time.
1 Denaturation. The first step in a PCR is the thermal denaturation of the double-stranded DNA template to separate the strands. This is achieved by raising the temperature of a reaction mixture to 95°C. The reaction mixture is comprised of the sample DNA that contains the target DNA to be amplified, a vast molar excess of the two oligonucleotide primers, a thermostable DNA polymerase (e.g., Taq DNA polymerase, isolated from the bacterium Thermus aquaticus), four deoxyribonucleotides, and the reaction buffer.
2 Annealing. For the second step, the temperature of the mixture is slowly cooled. During this step, the primers base-pair, or anneal, with their complementary sequences in the DNA template. The temperature at which this step of the reaction is performed is determined by the nucleotide sequence of the primer that forms hydrogen bonds with complementary nucleotides in the target DNA. Typical annealing temperatures are in the range of 45 to 68°C, although optimization is often required to achieve the desired outcome, that is, a product consisting of fragments of target DNA sequence only.
3 Extension. In the third step, the temperature is raised to ∼70°C, which is optimal for the catalytic activity of Taq DNA polymerase. DNA synthesis is initiated at the 3′ hydroxyl end of each annealed primer, and nucleotides are added to extend the complementary strand using the sample DNA as a template.
To understand how the PCR protocol succeeds in amplifying a discrete segment of DNA, it is important to keep in mind the location of each primer annealing site and its complementary sequence within the strands that are synthesized during each cycle. During the extension phase of the first cycle, the newly synthesized DNA from each primer is extended beyond the endpoint of the sequence that is complementary to the second primer. These new strands form “long templates” that are used in the second cycle (Fig. 2.20).
Figure 2.20 PCR. During a PCR cycle, the template DNA is denatured by heating and then slowly cooled to enable two primers (P1 and P2) to anneal to complementary (black) bases flanking the target DNA. The temperature is raised to about 70°C, and in the presence of the four deoxyribonucleotides, Taq DNA polymerase catalyzes the synthesis of a DNA strand extending from the 3′ hydroxyl end of each primer. In the first PCR cycle, DNA synthesis continues past the region of the template DNA strand that is complementary to the other primer sequence. The products of this reaction are two long strands of DNA that serve as templates for DNA synthesis during the second PCR cycle. In the second cycle, the primers hybridize to complementary regions in both the original strands and the long template strands, and DNA synthesis produces more long DNA strands from the original strands and short strands from the long template strands. A short template strand has a primer sequence at one end and the sequence complementary to the other primer at its other end. During the third PCR cycle, the primers hybridize to complementary regions of original, long template, and short template strands, and DNA synthesis produces long strands from the original strands and short strands from both long and short templates. By the end of the 30th PCR cycle, the products (amplicons) consist predominantly of short double-stranded DNA molecules that carry the target DNA sequence delineated by the primer sequences. Note that in the figure, newly synthesized strands are differentiated from template strands by a terminal arrow.
During the second cycle, the original sample DNA strands and the new strands synthesized in the first cycle (long templates) are denatured and then hybridized with the primers. The large molar excess of primers in the reaction mixture ensures that they will hybridize to the template DNA before complementary template strands have the chance to reanneal to each other. A second round of synthesis produces long templates from the original strands as well as some DNA strands that have a primer sequence at one end and a sequence complementary to the other primer at the other end (“short templates”) from the long templates (Fig. 2.20).
During the third cycle, short templates, long templates, and original strands all hybridize with the primers and are replicated (Fig. 2.20). In subsequent cycles, the short templates accumulate, and by the 30th cycle, these molecules, which are the desired PCR product, are about a million times more abundant than either the original or long template strands.
The specificity, sensitivity, and simplicity of PCR have rendered it a powerful technique that is central to many applications in molecular biotechnology, as illustrated throughout this book. For example, it is used to obtain large amounts of insert DNA for cloning, to detect specific mutations that cause genetic disease, to confirm biological relatives, to identify individuals suspected of committing a crime, and to diagnose infectious diseases (see chapter 4). Specific viral, bacterial, or fungal pathogens can be detected in samples from infected patients containing complex microbial communities by utilizing PCR primers that anneal to a sequence that is uniquely present in the genome of the pathogen. This technique is often powerful enough to discriminate among very similar strains of the same species of pathogenic microorganisms, which can assist in epidemiological investigations.
Cloning PCR Products
PCR is commonly used to amplify target DNA for cloning into a vector. To facilitate the cloning process, restriction enzyme recognition sites are added to the 5′ end of each of the primers that are complementary to sequences that flank the target sequence in a genome (Fig. 2.21). This is especially useful when suitable restriction sites are not available in the regions flanking the target DNA. Although the end of the primer containing the restriction enzyme recognition site lacks complementarity and therefore does not anneal to the target sequence, it does not interfere with DNA synthesis. Base-pairing between the 20 or so complementary nucleotides at the