Gene Sequencing: Technological Insights and Its Profound Impact on Humanity

DNA polymerase plays a crucial role in molecular diagnostics, which is mainly reflected in the following aspects:

• Polymerase Chain Reaction (PCR)
  • DNA Amplification: PCR is a commonly used technique in molecular diagnostics for amplifying the quantity of specific DNA fragments to make them detectable and analyzable. DNA polymerase is the core enzyme in the PCR reaction. It uses single-stranded DNA as a template and, according to the principle of base complementary pairing, adds free deoxynucleotides one by one to the 3′-OH end of the primer to synthesize a new DNA strand complementary to the template strand, achieving exponential amplification of the DNA fragment. Through PCR, trace amounts of DNA samples can be amplified to a detectable level, enhancing the sensitivity of detection.
  • Ensuring Amplification Accuracy: High-fidelity DNA polymerase has 3’→5′ exonuclease activity, which can correct misincorporated nucleotides during DNA synthesis. It can timely excise incorrectly paired bases and add the correct bases, thereby reducing the error rate during PCR amplification and ensuring the accuracy of the amplified product, providing a reliable DNA sample for subsequent diagnostic analysis.
• Quantitative Fluorescent PCR (qPCR)
  • Quantitative Analysis: In qPCR, DNA polymerase is also responsible for DNA amplification. By adding a fluorescent group to the PCR reaction system, the PCR amplification process can be monitored in real-time using the change in the fluorescent signal. While DNA polymerase amplifies the DNA, the fluorescent signal increases as the number of DNA copies increases. According to the cycle number (Ct value) when the fluorescent signal reaches the set threshold, the starting template DNA can be quantitatively analyzed. This is of great significance for pathogen load monitoring, quantitative analysis of gene expression, etc., and is helpful for disease diagnosis, treatment monitoring, and prognosis evaluation.
• Gene Sequencing
  • DNA Synthesis for Sequencing Reactions: In the Sanger sequencing method, DNA polymerase is used to synthesize a new strand complementary to the template DNA. In addition to normal deoxynucleotides, a small amount of fluorescently labeled dideoxynucleotides (ddNTPs) are added to the reaction system. When ddNTP is randomly incorporated into the DNA strand being synthesized, it terminates the extension of the DNA strand, generating a series of DNA fragments of different lengths. These fragments are separated by electrophoresis and detected by fluorescence, enabling the determination of the DNA sequence. In next-generation sequencing technologies, such as second-generation sequencing technologies based on the principle of sequencing by synthesis, DNA polymerase is also a key enzyme for DNA synthesis and sequencing, ensuring the accurate addition of nucleotides in each cycle and determining the DNA sequence by detecting the incorporation of nucleotides.
• Genotyping
  • Allele-Specific Amplification: In some genotyping methods, the characteristics of DNA polymerase are utilized for allele-specific amplification. By designing specific primers that are complementary to the specific bases of the target allele at the 3′ end, DNA polymerase can effectively extend the primers only when the primers match the template completely. In this way, different alleles can be distinguished, which is used for genotyping of disease-related genes, pharmacogenomics research, etc., helping doctors develop personalized treatment plans according to the genetic characteristics of patients.
• Nucleic Acid Molecular Hybridization
  • Probe Labeling: In nucleic acid molecular hybridization technology, DNA probes need to be labeled for detection. DNA polymerase can be used to label probes by methods such as nick translation or random primer method. For example, in the nick translation method, DNase I is first used to generate some single-stranded nicks in the DNA probe. Then, DNA polymerase I uses its 5’→3′ exonuclease activity to excise a segment of DNA at the nick and, at the same time, uses its 5’→3′ polymerase activity to add labeled deoxynucleotides to the 3′ end of the nick, thus preparing a labeled DNA probe for detecting specific nucleic acid sequences.