Polymerase chain reaction is a molecular biology technology used to amplify specific DNA fragments. It can be regarded as special DNA replication outside the organism. The biggest feature of PCR is that it can Trace amounts of DNA are greatly increased. Therefore, whether it is ancient creatures in fossils, the remains of historical figures, or the hair, skin or blood left by the murderer in a murder case decades ago, as long as a little bit of DNA can be isolated, it can be amplified by PCR and compared. right. This is where the power of “trace evidence” lies. Mullis from the United States first proposed the idea in 1983, and in 1985 he invented the polymerase chain reaction, a simple DNA amplification method, which meant the true birth of PCR technology. As of 2013, PCR has developed into the third generation technology. In 1973, Taiwanese scientist Qian Jiayun discovered the stable Taq DNA polymerase and made a fundamental contribution to the development of PCR technology.
PCR (polymerase chain reaction) uses DNA to denature into a single strand at a high temperature of 95°C in vitro. At low temperature (usually around 60°C), the primers are complementary to the single strand based on their bases. The principle of pairing is combined, and then the temperature is adjusted to the optimal reaction temperature of DNA polymerase (around 72°C). DNA polymerase synthesizes complementary strands along the direction from phosphate to five-carbon sugar (5'-3'). A PCR machine based on polymerase is actually a temperature control device that can control the denaturation temperature, renaturation temperature, and extension temperature well.
Chinese name
Polymerase chain reaction
Foreign name
Polymerase Chain Reaction
Abbreviation
p>
PCR
Attributes
Chain reaction
Creation of PCR
Khorana (1971) and others were the first to propose nucleic acid in vitro The idea of ??amplification: "After DNA is denatured, hybridized with a suitable primer, the primer is extended with a DNA polymerase, and the tRNA gene can be synthesized by repeating the process."
However, because gene sequence analysis methods were not yet available at that time With a mature, thermostable DNA polymerase yet to be reported and difficulties with primer synthesis, this idea seems moot. In addition, the emergence of molecular cloning technology in the early 1970s provided a way to clone and amplify genes, so Khorana's idea was forgotten.
In 1985, Kary Mullis invented PCR while working at Cetus. Mullis needs to synthesize DNA primers for sequencing work, but is often troubled by not having enough template DNA.
On a Friday night in April 1983, while driving to his country house, the idea of ??"polymerase chain reaction" suddenly occurred to him.
In December 1983, Mullis used isotope labeling to see the first PCR fragment of 49 bp in length after 10 cycles;
Applying for PCR on October 25, 1985 The patent was approved on July 28, 1987 (patent number 4,683,202), and Mullis was the first inventor;
The first academic paper on PCR was published in Science magazine on December 20, 1985. Mullis is ***'s co-author;
In May 1986, Mullis gave a special report at Cold Spring Harbor Laboratory, and the whole world began to learn the PCR method. [1]
PCR principle
Semi-conservative replication of DNA is an important way of biological evolution and generation. Double-stranded DNA can be denatured and unwinded into single strands under the action of various enzymes. With the participation of DNA polymerase, it is copied into two identical molecular copies according to the principle of complementary base pairing. In experiments, it was found that DNA can denature and melt at high temperatures, and can renature into double strands when the temperature is lowered. Therefore, by controlling the denaturation and renaturation of DNA through temperature changes, adding designed primers, DNA polymerase, and dNTPs, the in vitro replication of specific genes can be completed.
However, DNA polymerase will be inactive at high temperatures. Therefore, new DNA polymerase must be added for each cycle, which is not only cumbersome to operate, but also expensive, which restricts the application and development of PCR technology.
The discovery of the thermostable DNA polymerase-Taq enzyme is a milestone for the application of PCR. The enzyme can withstand high temperatures above 90°C without becoming inactive. There is no need to add enzyme in each cycle. This makes PCR technology very simple and greatly reduces the cost. PCR technology has been widely used and gradually applied in clinical practice.