HCY™ Pfu DNA Polymerase 

I. Definition and Basic Introduction

DNA Polymerase, namely DNA polymerase, is an enzyme that plays a crucial role in processes such as DNA replication, repair, and recombination. It uses DNA as a template and adds deoxyribonucleotides (dNTPs) one by one to the 3′-OH end of the DNA strand being synthesized, following the principle of base complementary pairing (A – T, C – G) to synthesize a new DNA strand.

II. Main Types and Characteristics

1. DNA Polymerases in Prokaryotes

• Taking Escherichia coli as an example, it contains multiple DNA polymerases. Among them, DNA polymerase I has been studied quite thoroughly. It has 5’→3′ polymerase activity and can synthesize a new DNA strand under the guidance of the template strand. Meanwhile, it also has 5’→3′ exonuclease activity and 3’→5′ exonuclease activity. The 5’→3′ exonuclease activity can be used to remove RNA primers. During DNA replication, RNA primers initiate the synthesis of DNA strands, and then DNA polymerase I will remove the RNA primers and fill in the corresponding DNA sequences. The 3’→5′ exonuclease activity plays a proofreading role. When incorrect base pairing occurs in the newly synthesized DNA strand, it can remove the incorrect nucleotides to ensure the accuracy of DNA replication.
• DNA polymerase III is the main replicative enzyme in Escherichia coli. It has a high polymerase activity and can synthesize DNA strands at a relatively fast speed. It also has 3’→5′ exonuclease activity to ensure the accuracy of replication.

2. DNA Polymerases in Eukaryotes

• There are multiple DNA polymerases in eukaryotes, such as DNA polymerase α, β, γ, δ, and ε. DNA polymerase α is mainly involved in initiating DNA replication. It combines with primase and synthesizes an RNA-DNA primer at the origin of replication, providing an initiation site for the subsequent synthesis of DNA strands. DNA polymerase δ and ε are mainly responsible for the elongation of DNA strands. They continuously add dNTPs along the template strand during DNA replication to elongate the DNA strand. DNA polymerase β is mainly involved in the DNA repair process. When DNA is damaged, it can repair the damaged sites to ensure the integrity of the DNA structure. DNA polymerase γ mainly exists in mitochondria and is responsible for the replication and repair of mitochondrial DNA.

III. Functions and Applications

1. Function in DNA Replication

• During cell division, DNA polymerase is the core enzyme for DNA replication. It can accurately replicate the genetic information of parental DNA, enabling daughter cells to obtain the same DNA as parental cells. For example, during the division of human cells, DNA polymerase synthesizes a new DNA strand along the single-stranded template after the double-stranded DNA of parental cells is unwound, following the principle of base complementary pairing, at a speed of about 50 – 100 nucleotides per second, ensuring the stable transmission of genetic information.

2. Application in DNA Repair

• DNA polymerase plays a key role in DNA damage repair. When DNA is damaged by factors such as ultraviolet light and chemical substances, such as base deletion, mismatch, or double-strand breaks, DNA polymerase can identify the damaged sites and use its polymerase and exonuclease activities for repair. For example, during base excision repair, first, specific glycosylases remove the damaged bases, and then DNA polymerase adds the correct bases at the gaps. Finally, DNA ligase connects the repaired fragments to restore the normal structure of DNA.

3. Applications in Molecular Biology Techniques

• In the polymerase chain reaction (PCR) technique, DNA polymerase is an essential component. PCR is a technique used for in vitro amplification of specific DNA fragments. Through the cycles of high-temperature denaturation (unwinding of DNA double strands), low-temperature annealing (primers binding to the template), and appropriate-temperature elongation (DNA polymerase synthesizing new strands), the target DNA fragments can be amplified in large quantities. The commonly used Taq DNA polymerase is isolated from thermophilic bacteria. It has the characteristic of being heat-resistant and can still maintain its activity after the high-temperature denaturation step in the PCR process, thus achieving efficient amplification of DNA. This technique has wide applications in many fields such as gene cloning, gene diagnosis, and forensic identification.

IV. Influencing Factors and Precautions

1. Effect of Temperature

• Different types of DNA polymerases have their appropriate temperature ranges. For example, the optimal temperature for Taq DNA polymerase is around 72 °C. At this temperature, its polymerase activity is the highest, and it can quickly synthesize DNA strands. If the temperature is too high or too low, its activity will be affected. Excessively high temperature may lead to the denaturation and inactivation of the enzyme, while too low temperature will reduce the enzyme’s activity, prolong the time for DNA synthesis or even result in incomplete synthesis.

2. Quality of Template and Primers

• The action of DNA polymerase depends on high-quality DNA templates and appropriate primers. The template DNA should have a complete sequence structure and avoid serious damage or breaks. The design of primers should comply with the principle of base complementary pairing, and the length and GC content should be appropriate. If the primers are improperly designed, such as being too short or too long, or having too high or too low GC content, it may lead to low binding efficiency between the primers and the template, affect the working efficiency of DNA polymerase, or even make it impossible to carry out effective DNA synthesis.

3. Ion Concentration in the Reaction System

• The concentration of magnesium ions (Mg²⁺) in the reaction system has an important impact on the activity of DNA polymerase. Magnesium ions are activators of DNA polymerase. They can bind to the enzyme and the substrate (dNTPs) and promote the interaction between the enzyme and the substrate. If the concentration of magnesium ions is too high, it may lead to non-specific amplification or inhibition of enzyme activity. If the concentration of magnesium ions is too low, the enzyme’s activity will be insufficient, affecting DNA synthesis. Therefore, during the experiment, it is necessary to precisely control the concentration of magnesium ions to ensure the optimal working state of DNA polymerase.

As an important biological enzyme, DNA polymerase plays an irreplaceable role in maintaining the stable transmission of genetic information and various biochemical processes. Its research and application have far-reaching significance for the development of multiple fields such as biology and medicine.