The initiation of the synthesis of two new strands complementary to the original DNA strands occurs by adding DNA pol and dNTP (in excess) to the mixture at approximately 72°C. DNA pol adds nucleotides to the 3′-hydroxyl end of the primer, leading to strand growth in the 5′→3′ direction across the target DNA. This results in the creation of complementary copies of the target DNA. It should be noted that the PCR products can have a length of several thousand base pairs. After one cycle of replication is completed, the reaction mixture is heated to separate the strands, resulting in a total of four strands. Each strand then binds a complementary primer, and the process of primer extension is repeated. The use of a heat-stable DNA pol, such as Taq from the bacterium Thermus aquaticus, eliminates the need to add the polymerase at each cycle, as it does not denature. However, Taq lacks proofreading activity. Usually, 20–30 cycles are conducted during this process, leading to a substantial amplification of the DNA, ranging from a million-fold to a billion-fold. Each extension product contains a sequence at its 5′-end that is complementary to the primer, enabling each newly synthesized strand to act as a template for the successive cycles. This exponential increase in the amount of target DNA with each cycle gives the technique its name, "polymerase chain reaction." Probes can be generated during PCR by incorporating labeled nucleotides in the final few cycles.

PCR offers several advantages over biologic cloning in terms of amplifying a specific DNA sequence. It provides increased sensitivity and speed. PCR can amplify DNA sequences that are present in trace amounts and make them the predominant sequence. The technique is so sensitive that it allows the amplification and study of DNA sequences present in a single cell. When compared to traditional cloning methods using recombinant DNA techniques, isolating and amplifying a specific DNA sequence through PCR is faster and less technically challenging.

PCR has become a widely used tool in various fields, including research, forensics, and clinical diagnostics. Its applications are diverse:
1. Comparing a normal gene to its mutant form: PCR enables the synthesis of mutant DNA in sufficient quantities for sequencing without the need for laborious biologic cloning of the DNA.
2. Forensic analysis of DNA samples: PCR-based DNA fingerprinting has revolutionized the analysis of evidence from crime scenes. It only requires a small amount of DNA isolated from sources like a single human hair, a tiny blood spot, or a sample of semen to determine whether it belongs to a specific individual. The DNA markers used for such fingerprinting are typically short tandem repeats, which are smaller in size than the previously described VNTR.
3. Detecting low-abundance nucleic acid sequences: Conventional methods often struggle to detect viruses with long latency periods, such as human immunodeficiency virus (HIV), during the early stages of infection. PCR provides a rapid and sensitive means of detecting viral DNA sequences, even when the virus is present in a small proportion of cells. Quantitative PCR (qPCR), also known as real-time PCR, allows for the quantification of the target nucleic acid's amount (copy number) after each cycle of amplification, making it useful in determining viral load (the amount of virus).
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Extending primers: DNA pol and dNTP (in excess) are added to the mixture (~72°C) to initiate the synthesis of two new strands complementary to the original DNA strands. DNA pol adds nucleotides to the 3′-hydroxyl end of the primer, and strand growth extends in the 5′→3′ direction across the target DNA, making complementary copies of the target. [Note: PCR products can be several thousand base pairs long.] At the completion of one cycle of replication, the reaction mixture is heated again to separate the strands (of which there are now four). Each strand binds a complementary primer, and the step of primer extension is repeated. By using a heat-stable DNA pol (for example, Taq from the bacterium Thermus aquaticus that normally lives at high temperatures), the polymerase is not denatured and, therefore, does not have to be added at each successive cycle. However, Taq lacks proofreading activity. Typically, 20–30 cycles are run during this process, amplifying the DNA by a million-fold (220) to a billion-fold (230). [Note: Each extension product includes a sequence at its 5′-end that is complementary to the primer (see Fig. 34.20). Thus, each newly synthesized strand can act as a template for the successive cycles (see Fig. 34.21). This leads to an exponential increase in the amount of target DNA with each cycle, hence, the name “polymerase chain reaction.”] Probes can be made during PCR by adding labeled nucleotides to the last few cycles.
B. Advantages
The major advantages of PCR over biologic cloning as a mechanism for amplifying a specific DNA sequence are sensitivity and speed. DNA sequences present in only trace amounts can be amplified to become the predominant sequence. PCR is so sensitive that DNA sequences present in an individual cell can be amplified and studied. Isolating and amplifying a specific DNA sequence by PCR is faster and less technically difficult than traditional cloning methods using recombinant DNA techniques.
C. Applications
PCR has become a very common tool in research, forensics, and clinical diagnostics.
1.
Comparison of a normal gene to its mutant form: PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laborious biologic cloning of the DNA.
2.
Forensic analysis of DNA samples: DNA fingerprinting by means of PCR has revolutionized the analysis of evidence from crime scenes. DNA isolated from a single human hair, a tiny spot of blood, or a sample of semen is sufficient to determine whether the sample comes from a specific individual. The DNA markers analyzed for such fingerprinting are most commonly a type of polymorphism known as short tandem repeats. These are very similar to the VNTR described previously (see p.
491) but are smaller in size. [Note: Paternity testing uses the same techniques.] 3.
Detection of low-abundance nucleic acid sequences: Viruses that have a long latency period, such as human immunodeficiency virus (HIV), are difficult to detect at the early stage of infection using conventional methods. PCR offers a rapid and sensitive method for detecting viral DNA sequences even when only a small proportion of cells harbors the virus. [Note: Quantitative PCR (qPCR), also known as real-time PCR, allows quantification of the amount (copy number) of the target nucleic acid after each cycle of amplification (that is, in real time) rather than at the end and is useful in determining viral load (the amount of virus).] 4.