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The replicator of life: Why is thermostable DNA polymerase the star in PCR?
In the fields of genetic engineering and molecular biology, the emergence of thermostable DNA polymerase is undoubtedly a revolutionary breakthrough. These enzymes from Rexi Biotechnology have enabled the rapid evolution of polymerase chain reaction (PCR) technology and are widely used in gene replication, gene therapy and many other biotechnology fields.
Thermostable DNA polymerases have naturally selected properties that allow them to remain functional at high temperatures, eliminating the need for frequent additions of enzymes in PCR.
Characteristics of thermostable DNA polymerase
Thermostable DNA polymerases are usually derived from thermophilic bacteria or archaea, and these enzymes have excellent activity at high temperatures. Most of these polymerases possess 5'→3' polymerization activity and possess 5'→3' or 3'→5' exonuclease activity.
Its structure and function
The structure of these polymerases is shaped like a hand, with a thumb, palm and fingers. The function of the thumb is to bind and move double-stranded DNA, the palm carries the active center of the polymerase, and the fingers are responsible for binding substrates, such as template DNA and nucleotide triphosphates.
Polymerases from different sources
Among bacterial thermostable DNA polymerases, Taq enzyme is widely used due to its excellent performance. In addition, there are polymerases such as Tfl, Tma, Tne, Tth and Bst. In contrast, archaeal polymerases include Pfu, Pwo, etc. Most of these polymerases have the ability to correct synthetic errors, that is, 3' → 5' exonuclease activity.
A mixture of archaeal and bacterial polymerases has been shown to efficiently synthesize DNA fragments up to 35 kb in longest range PCR.
The speed and processing power of polymerase
The synthesis rates of different polymerases vary greatly. For example, Taq polymerase has a synthesis rate of 60 nucleotides per second, while KOD polymerase can achieve 120 nucleotides per second. These properties affect reaction efficiency and yield in PCR applications.
Error rate and yield
Error rate is an important indicator for evaluating polymerase quality. Taq polymerase has an error rate of approximately 8 errors per 1,000 nucleotides, while Pfu polymerase has an error rate as low as less than 1 error. Generally speaking, bacterial polymerases produce higher yields but are accompanied by more replication errors, while archaeal polymerases produce less but purer DNA.
Application fields
In addition to its application in PCR technology, thermostable DNA polymerases have also shown their importance in many branches of biology, including RNA transcription, quantitative PCR (QPCR), on-demand mutagenesis, and DNA sequencing. These techniques help scientists gain a deeper understanding of the fundamental ingredients of life and how it works.
Historical background
In 1976, Alice Chien first characterized the thermostable Taq polymerase, and in 1988, Randall K. Saiki introduced it into PCR technology, marking a major change in gene replication technology. In the following years, the gene cloning and improvement of polymerase and the application of various high-efficiency PCR technologies continued to advance.
As time goes by, more and more research will focus on how to further improve the performance of thermostable DNA polymerases to meet the growing scientific needs.
However, with the development of thermostable DNA polymerases, new challenges are constantly emerging, such as how to further improve their accuracy and efficiency in gene editing and synthetic biology. These questions make people think about how future polymerases will face these challenges.
