PCR (Polymerase Chain Reaction) is a fundamental molecular biology technique used to exponentially amplify target sequences from a DNA template. It allows for the rapid and efficient copying of specific DNA fragments. The process of PCR is divided into three main steps: denaturation, annealing (or hybridization), and extension. These three steps are repeated in cycles, enabling the rapid amplification of DNA.

1. Denaturation

During the denaturation step, the template DNA is heated to a high temperature (usually 94-98°C), which is sufficient to break the hydrogen bonds between the double-stranded DNA, resulting in the separation of the strands into single strands. This step is the starting point of the PCR cycle and provides the necessary single-stranded template for subsequent DNA synthesis.

2. Annealing

The temperature is then rapidly lowered to a lower level (typically 50-65°C). At this temperature, primers can specifically bind to the single-stranded regions of the template DNA. The design of the primers is crucial; they must perfectly match specific regions of the target DNA sequence to ensure the accuracy of DNA synthesis. The binding of the primers provides a clear starting point for DNA polymerase, thereby initiating the synthesis of new DNA strands.

3. Extension

The temperature is raised again to a level suitable for the activity of DNA polymerase (usually 72°C). At this temperature, DNA polymerase moves along the single-stranded template DNA, using dNTPs as building blocks to synthesize new DNA strands according to the principle of base complementary pairing. In this process, DNA polymerase begins synthesis from the 3' end of the primer until it reaches the end of the template DNA or encounters the next primer binding site. Thus, each newly synthesized DNA molecule contains one original template strand and one newly synthesized complementary strand, achieving semi-conservative replication of DNA.

The three steps of the PCR cycle are repeated multiple times (typically 20-40 cycles), with each cycle doubling the amount of DNA, resulting in exponential amplification. This allows even trace amounts of DNA template to be replicated in large quantities within a short period, showcasing PCR technology's high sensitivity and efficiency. Furthermore, the specificity of PCR is ensured by the precise design of the primers and the fidelity of DNA polymerase, making it a highly reliable and precise method for DNA amplification.

In summary, the PCR technique cleverly utilizes template DNA, primers, DNA polymerase, and dNTPs to achieve rapid, efficient, and specific amplification of DNA in a laboratory setting.

Synbio Technologies | PCR subcloning

Synbio Technologies, leveraging the integration of DNA synthesis tools and cloning technologies, offers comprehensive PCR cloning and subcloning services. Our services are capable of cloning the target gene into any desired location of the vector, fulfilling your specific design requirements. For subcloning the same gene fragment into different vectors, we also provide efficient services to ensure the smooth progress of your downstream experiments.

PCR cloning and subcloning workflow