What is PCR ?
The full name of PCR is short for Polymerase Chain Reaction. Biology can be divided into two eras: one without PCR and one with PCR. Without PCR (Polymerase Chain Reaction) technology, modern molecular biology would not exist. Dr. Kary Mullis, the inventor of PCR, was awarded the Nobel Prize in Chemistry in 1993 and is hailed as the father of PCR worldwide.
The genetic information of organisms is stored within DNA molecules, but analyzing this genetic information requires a large amount of DNA. In 1985, Dr. Kary Mullis invented an efficient method that could rapidly amplify a small amount of DNA. By heating, the two strands of the DNA molecule are separated, and added DNA primers bind to each strand. With the enzyme DNA polymerase, new DNA strands can be synthesized, and the process can be repeated. PCR has significant implications in medical research and forensic science.
What PCR can do?
PCR is a powerful technique that allows for the efficient replication of DNA through a simple and effective method. It can be considered as a specialized DNA replication process outside of living organisms. Through PCR technology, a large amount of DNA can be amplified from a minimal starting material in a short period of time. This enables scientists to conduct various research and applications, including gene sequencing, gene mutation analysis, gene expression studies, disease diagnosis and monitoring, and more.
What is the principle of PCR?
The basic principle of PCR is to exponentially amplify the target DNA sequence through repeated cycles of DNA denaturation, primer annealing, and DNA synthesis. This amplification process can be completed in a short period of time and allows for the detection of the target DNA sequence from a minimal starting material. The characteristics of PCR technology include high sensitivity, high specificity, and fast reaction speed.
What are the PCR reaction steps?
The standard PCR process consists of three steps:
1. Denaturation (90°C-96°C): The double-stranded DNA template is exposed to high temperature, causing the hydrogen bonds to break and resulting in single-stranded DNA.
2. Annealing (60°C-65°C): The temperature is lowered, allowing the primers to bind to the DNA template, forming localized double-stranded regions.
3. Extension (70°C-75°C): With the action of Taq polymerase (optimal activity around 72°C), using dNTPs as building blocks, DNA synthesis occurs from the 3' end of the primers in a 5' to 3' direction, resulting in the synthesis of a complementary DNA strand to the template. Each cycle of denaturation, annealing, and extension leads to a doubling of the DNA content.
As shown in the diagram, some PCR reactions with short amplification regions can be completed even when Taq polymerase is not at its optimal activity. In such cases, a two-step PCR can be employed, where annealing and extension are performed simultaneously at a temperature range of 60°C.
What are the advantages of PCR ?
High Specificity:
The specificity of PCR reaction is determined by:
- Specific and correct binding of primers to the target DNA.
- Principles of base pairing.
- Faithfulness of Taq DNA polymerase in synthesizing the reaction.
- Specificity and conservation of the target gene.
High Sensitivity:
PCR is highly sensitive and can detect very low amounts of target DNA or RNA.
Convenience and Speed:
PCR is a relatively fast and convenient technique, providing rapid amplification of DNA or RNA sequences.
Low Purity Requirement:
PCR can work with impure or partially degraded DNA samples, making it suitable for various sample types.
In medical research, PCR technology is widely used in disease diagnosis, genetic disease screening, pathogen detection, tumor gene testing, and more. In forensic science, PCR technology is used for DNA fingerprinting to help solve criminal cases and perform paternity testing.
We will explore different types of the PCR in our next article, see you soon.
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