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The polymerase chain reaction (PCR) is essentially DNA replication in a test tube. In the laboratory, PCR can be used to copy or amplify any DNA sequence of interest. PCR has a myriad of applications. For example, PCR can be used in forensics to make copies of the DNA molecules left by a suspect at a crime scene, scientists can use PCR to make many copies of a gene to study gene structure and function, and finally, PCR can be used to determine if an individual is infected with a microbe, such as the human immunodeficiency virus (HIV) or SARS-CoV-2 (the virus that causes COVID-19).
Suppose our goal is to study the human insulin gene. We could use PCR to make millions of copies of the insulin gene as part of our research. To accomplish this goal, our PCR reaction would contain the following components:
Once the PCR reaction is prepared containing each of the five components above, the PCR reaction is subjected to multiple PCR cycles (see figure 8.1). Each PCR cycle has the following three steps:
Each PCR cycle described above is repeated typically 30-35 times. The number of DNA copies (i.e., the number of copies of the human insulin gene) is doubled at the conclusion of each cycle. The total number of template (target) DNA copies made during PCR can be estimated using the following equation:
n = a x 2b
In the lab, the PCR cycles are accomplished by mixing the above five PCR reaction components in a test tube, followed by placing the PCR reaction in a thermocycler device. The thermocycler automates the number of cycles, the temperature of each step within a cycle, and the length of each step within a cycle.
Agarose gel electrophoresis is used in the laboratory to visualize DNA. For our purposes, agarose gel electrophoresis can be used to determine whether the PCR experiment successfully copied (amplified) the human insulin gene. Since agarose gel electorphoresis separates DNA molecules from each other based upon size, agarose gel electrophoresis can also be used to analyze the size (in base pairs) of the insulin PCR products (see Figure 8.2).
To perform an agarose gel electrophoresis experiment:
If PCR amplification of the insulin gene was successful, a band will be seen in the agarose gel when exposed to ultraviolet light.
PCR can also be used to amplify a particular mRNA molecule; however, mRNAs are not used directly as templates for the PCR reaction. To amplify a mRNA molecule, reverse transcription is done prior to PCR. Reverse transcription converts the mRNA molecules within a cell into a collection of DNA molecules called complementary DNAs (cDNAs). These cDNAs can then be used as templates in PCR.
Reverse transcription to produce cDNAs involves the following steps:
Reverse transcription produces a collection of double-stranded cDNA molecule that correspond to the entire mRNA collection within a cell. This collection of various cDNA molecules can then serve as the templates for PCR. Since the primers used in PCR are typically designed to be specific for a particular gene sequence, PCR amplification copies of a single cDNA type, corresponding to a single type of mRNA. In essence, this reverse transcription PCR process has amplified a single type of mRNA from the cell.
Insert a figure here that illustrates reverse transcription PCR.
Often, the goal of PCR is to make many copies of a particular DNA sequence. The success of the PCR experiment is determined by analyzing the PCR product on an agarose gel, as described above. In other cases, the goal of PCR is to determine how many copies of the template DNA molecule are present at the beginning of the PCR experiment, before PCR amplification occurs. To determine the number of template DNA molecules present in a sample, a modification of PCR, called real-time PCR or quantitative PCR (qPCR) is used.
Real-time PCR allows a scientist to monitor the production of PCR products in real-time (i.e., as the reaction is occurring in the thermocycler). If the concentration of template DNA molecules in the reaction is low prior to the start of PCR, then it takes more PCR cycles to produce the number of PCR products required for detection. Alternatively, if the concentration of template DNA molecules in the reaction is high prior to the start of PCR, then detectable products are formed in earlier PCR cycles. Thus, the real-time PCR technique is especially powerful, as it allows researchers to quantitatively measure the concentration of template DNA sequences. Moreover, if the template molecules are cDNA molecules produced from mRNA by reverse transcription, then real-time PCR can provide a quantitative measure of how actively a gene is transcribed (the more active the gene, the more mRNA molecules are produced by the gene).
Real-time PCR involves a modification to the conventional PCR approach. In addition to the five PCR components described earlier, a real-time PCR reaction contains a probe molecule. A common probe that is used in many real-time PCR reactions is TaqMan. The TaqMan probe is a short DNA molecule that is designed to form hydrogen bonds to one of the two template DNA strands downstream (in the 3' direction) from one of the two DNA primers. The 5’ and 3’ ends of the TaqMan molecule have been modified; the 5’ end of TaqMan is attached to a fluorescent reporter molecule, while the 3’ end of TaqMan is attached to a quencher molecule. When the reporter and the quencher are in close proximity (i.e., within the same probe molecule), the quencher molecule inhibits the fluorescence produced by the reporter molecule. When the reporter molecule is separated from the quencher molecule (i.e., when the probe is degraded by the 5' to 3' exonuclease activity of Taq DNA polymerase), fluorescence occurs.
During the primer annealing step in a real-time PCR reaction, both a primer and the TaqMan probe binds to one of the two template DNA strands. During the DNA synthesis step, Taq polymerase synthesizes the daughter DNA strand toward the TaqMan probe bound to the template DNA strand. When Taq polymerase encounters the TaqMan probe, Taq polymerase uses its 5’ to 3’ exonuclease activity to begin digesting TaqMan. As the TaqMan probe is digested, the reporter molecule is separated from the quencher molecule and fluorescence occurs. If the number of template DNA molecules in the reaction is low, fluorescence is low in early PCR cycles and begins to be detectable in later PCR cycles. If the number template DNA molecules in the reaction is high, fluorescence is detectable in the earlier PCR cycles.
The thermocycler used in real-time PCR is modified to detect the fluorescence emitted by the reporter molecule as it is released from the quencher molecule during DNA replication.
Insert a figure here that illustrates a real-time PCR cycle.