Polymerase Chain Reaction (PCR) Methodology
The polymerase chain reaction (PCR) was originally developed in 1984 by the American biochemist Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his pioneering work. Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies (millions or billions!) of a particular region of DNA. This DNA region can be anything the experimenter is interested in. For example, it might be a gene whose function a researcher wants to understand, or a genetic marker used by forensic scientists to match crime scene DNA with suspects.
The goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments.
Since its development by Kary Mullis in 1984, PCR has revolutionized the molecular biology field, giving rise to many advantageous techniques that allow the analysis of different nucleic acids.
PCR is used in many areas of biology and medicine, including molecular biology research, medical diagnostics, and even some branches of ecology.
PCR Requirements
DNA Template
Primers
DNA POLYMERASE
dNTPs
Buffer/cofactors
Taq polymerase
Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates.
The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus). T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-stable and is most active around 70°C (a temperature at which a human or E. coli DNA polymerase would be non-functional).
This heat-stability makes Taq polymerase ideal for PCR. High temperature is used repeatedly in PCR to denature the template DNA, or separate its strands.
PCR Primers
Like other DNA polymerases, Taq polymerase can only make DNA if it's given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis.
n a PCR reaction, the experimenter determines the region of DNA that will be copied, or amplified, by the primers she or he chooses. PCR primers are short pieces of single-stranded DNA, usually around 18- 20 nucleotides in length.
Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied. The primers bind to the template by complementary base pairing.
When the primers are bound to the template, they can be extended by the polymerase, and the region that lies between them will get copied.
Cofactors and buffer
Magnesium (Mg2+) is an important cofactor for DNA polymerase. Magnesium assists phosphodiester bond formation and is required for successful PCR amplification. Mg2+ concentration should be maintained at around 0.5-5.0mM in reactions using Taq polymerase.
Usually, the concentration is higher than that of dNTPs (50μM of each is ideal) and primers, and must be optimized and determined by the template, buffer and dNTP content. In addition, some polymerases may require as high as 6mM Mg2+ while others need only ≤ 1mM Mg2+. Magnesium concentration can be changed to optimize PCR amplification.
Increasing magnesium concentration results in higher product yield. However, an increase in this cofactor also results in a decreased DNA polymerase specificity and fidelity.
Potassium salt (K+), present in the buffer, can also be modified to improve PCR amplification. Usually, in the buffer should be 35-100mM. It's important to note that amplification of a long PCR product may require a decrease in K+. The buffer in a PCR reaction serves to facilitate amplification by stabilizing the polymerase. Different PCR buffers are currently used, but the preferred one is Tris/HCl, which must maintain a pH of 8.4 at room temperature.
Steps In PCR
The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized.
The basic steps are:
Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2 - 4 hours, depending on the length of the DNA region being copied. If the reaction is efficient, the target region can go from just one or a few copies to billions.