Polymerase Chain Reaction (PCR) is a technique designed to amplify a specific segment of DNA exponentially. To achieve this replication, the reaction requires several components, including template DNA, primers, and a specialized enzyme. Deoxyribonucleoside triphosphates (dNTPs), commonly referred to as nucleotides, are added to the sample. They serve two distinct functions: acting as physical building materials and providing the energy source for synthesizing new DNA strands.
The Raw Materials of Replication
Deoxyribonucleoside triphosphates are the fundamental molecules that form the structure of DNA. These single units are linked together by the DNA polymerase enzyme to create the long DNA chain. The PCR mix must contain four specific dNTPs: deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP). These correspond to the four nitrogenous bases found in DNA: Adenine (A), Guanine (G), Cytosine (C), and Thymine (T).
Each dNTP molecule is composed of a deoxyribose sugar, a nitrogenous base, and a chain of three phosphate groups. The presence and proper balance of all four dNTPs are necessary to ensure the DNA polymerase accurately constructs the new strand, matching the template DNA through complementary base pairing.
Fueling the Polymerase Reaction
The primary function of dNTPs is to serve as the structural components that are incorporated into the growing DNA chain during the extension phase of PCR. The DNA polymerase enzyme moves along the single-stranded template, selecting the correct incoming dNTP that pairs with the base on the template. The polymerase then chemically links the new dNTP to the previous one on the chain, extending the new DNA segment in the 5’ to 3’ direction.
This process of linking the molecules together requires energy to form the strong phosphodiester bond that creates the DNA backbone. The dNTP molecule provides this energy through its three phosphate groups. The bond between the first (alpha) and second (beta) phosphate group is a high-energy bond.
When the DNA polymerase attaches the incoming nucleotide to the growing strand, it cleaves the bond between the alpha and beta phosphate groups. This cleavage releases the two outer phosphate groups (beta and gamma) as a molecule called pyrophosphate. The energy liberated from this hydrolysis reaction is harnessed by the polymerase to drive the formation of the new phosphodiester bond, allowing the chain to grow.
The dNTPs function as self-contained packets, supplying both the physical building block (the deoxyribonucleoside monophosphate) and the energy source (the cleavage of the triphosphate tail). This dual role allows the DNA polymerase to rapidly and repeatedly extend the new DNA strand during each cycle of the reaction.
The Practical Importance of Nucleotide Quality
The success of the PCR hinges on the correct concentration and high quality of the dNTP mixture. Typically, each of the four dNTPs is added to the reaction at an equal concentration, often around 0.2 millimolar (mM). If the concentration of any dNTP is too low, the polymerase may run out of the required building blocks, which can prematurely stop the synthesis and lead to poor or failed amplification.
Conversely, an excessively high concentration of dNTPs can also inhibit the reaction. The phosphate groups on the dNTPs bind to magnesium ions (\(\text{Mg}^{2+}\)), which are a required cofactor for the DNA polymerase enzyme to function. When dNTP levels are too high, they sequester too many \(\text{Mg}^{2+}\) ions, reducing the amount available for the polymerase and slowing its activity.
Furthermore, the purity of the dNTPs is a significant practical consideration. Contaminants, such as chemical inhibitors, can interfere with the polymerase activity, even if the dNTP concentration is optimal. High-quality dNTPs, free of impurities, ensure that the enzyme can perform its function efficiently and consistently across the many cycles of the reaction.