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Genentech (Genentech) launched a very high-profile radio program the previous year. It was called Two Scientists Walk into a Bar. The radio’s program covered the development of basic biology, chemistry, and cancer immune drugs. , clinical experimental design and other fields, is a rare high-quality popular science program.

The anchor of the radio is Jane Grogan, chief scientist of Genentech’s cancer immunology product development department, and the location of the show is at a bar. Grogan will invite scientists from all fields to discuss topics such as inflammation, pain, and cancer that are of high public concern. Grogan’s weird Australian accent, coupled with the background sounds in the bars, is perfect for my insomniacs who use hypnosis when they can’t sleep. Many people may wonder why Grogan chose the location of the show in a bar. This is actually to commemorate the meeting of Genenaker’s two founders, Boyer and Swanson.

Recombinant DNA technology

In the winter of 1968, Paul Berg ended his 11-month vacation and returned to Stanford University. Berg is a biochemist. He spent the first half of his career focusing on protein synthesis studies. However, during the 11 months of the sabbatical study at the Salk Institute, he began to rethink future research directions. . During this time, Berg and the Salk Institute virologist Renato Dulbecco worked on animal viruses. He spent a long time pondering over genes and viruses and wondering how genetic information was transmitted.

The structure of the virus is relatively simple. It usually contains only nucleic acids that carry genetic information and capsids that enclose nucleic acids. After the virus enters the host cell, it removes the outer shell and allows the genetic material to enter the host cell. The host cell is used as a factory for gene replication and expression to create new shells and genetic material, thereby generating millions of new viruses. Berg was very interested in one of these viruses: Monkey Virus 40 (SV40).

The genome size of SV40 is only 600,000 times that of the human genome. There are about 20,000 genes in humans and only 7 genes in SV40. Unlike many other viruses, SV40 can live in peace with many host cells. Some viruses can replicate their own cells and produce millions of new viruses that eventually lead to the death of the host cell. SV40 can insert its own DNA into the genome of the host cell (though this is not unique to SV40). If there is no specific activation signal, these genes inserted into the host cell genome will remain dormant.

Berg believes that SV40 is an ideal vehicle for transmitting human genetic information. He feels that if exogenous genes can be loaded into the SV40 genome, then this exogenous gene will likely be inserted into human cells along with the viral genome. In the genome, thus modifying the host cell’s genetic information. There is no doubt that if his vision can be realized, it will open a new chapter in the field of genetics.

Berg understands that achieving this goal is not an easy task. The first technical obstacle he faces is how to insert foreign genes into the genome of the virus, which means that he must find a way to artificially construct viral genes and foreign gene mosaics. .

Unlike the linear structure of DNA in human cells, the DNA of SV40 is a circular structure. After the virus enters the host cell, the circular DNA opens up to form a linear structure, and the viral DNA can be inserted into the host cell genome after the circular DNA is opened. Berg believes that if you want to insert an exogenous gene into the DNA of SV40, you need to first open the circular DNA of SV40, and then connect the ends of the DNA fragments together to re-form the circular DNA. The next task is to give the virus itself. Upon completion, the virus will carry the foreign gene to infect the host cell and insert the foreign DNA fragment into the genome of the host cell.

In fact, Berg is not the only one who has thought about how to open the virus circular DNA and insert foreign genes. In 1969, also at Stanford University, Peter Lobban, a graduate student in another research group, wrote a postgraduate mid-term assessment research program. He proposed similar genetic manipulations to modify the genetic information of another virus.

Lobban studied at the Massachusetts Institute of Technology (MIT) and influenced by MIT. He looks more like an engineer. Lobban wrote straightforwardly in the research plan that the genes in the cells are not different from the steel beams used in the building and can be modified to meet various needs of human beings, but the most important thing is how to find the tools for genetic modification. At that time, Lobban had started an experimental study under the direction of his mentor, Dale Kaiser, and tried to transfer genes from one DNA molecule to another using the enzymes commonly used in biochemical laboratories.

In fact both Berg and Lobban have realized that the SV40 should not be seen as a virus, but rather as a compound: all they need to do is insert a gene into the SV40 genome through a series of biochemical reactions. But Berg knew that he first needed to find an enzyme that could cleave circular DNA, and second was to get another enzyme that attached the DNA to the SV40 DNA.

But where to find these enzymes that can cut and link DNA? Perhaps looking for bacteria is a wise choice. Since the 1960s, researchers have begun to try to purify enzymes that can liberate free DNA in vitro from bacteria. Theoretically, this type of enzyme should be present in any cell, and the enzyme is involved in the process of cell replication (Okazaki fragment attachment) and the repair process of DNA damage.

DNA may break after injury, and cells need specific enzymes to reconnect broken DNA fragments during repair. Ligase is an enzyme that participates in the above process. It can reconnect two fragments formed by the cleavage of the DNA backbone, thus restoring the integrity of the DNA double helix structure.

However, DNA cleavage enzymes are not easily found. There is a demand for DNA ligase in almost all cells, but in general it is not necessary to cleave the DNA inside the cell. However, some organisms have such enzymes in their cells. Bacteria and viruses that grow in the harsh environment where nutrients are scarce have caused the bacteria to evolve a set of enzymes that can cleave invading viral DNA due to intense competition between them. , as a weapon of its own defense to resist the invasion of the virus (in fact, this is also the basis of part of the bacterial immune function, the adaptive immunity of another bacteria is the CRISPR system.

These bacteria can use DNA cleavage enzymes to cut the DNA of invading viruses. This enzyme is called a restriction enzyme (restriction means that they can recognize specific sequences of DNA and only cut DNA double helices at specific sites). , and this restriction is very important, if you can not achieve specific shear, such enzymes may affect their own DNA, resulting in DNA damage and induce apoptosis).

The above two enzymes are the basis of the Berg experiment. Berg is very clear about the application of this technology: genes can be recombined by recombination, these genes can be modified, he can also introduce mutant genes, can make genes transfer between different species; a frog’s genes can be inserted Into the viral genome, it is later introduced into the human genome. Human genes can also be introduced into the genome of bacteria in a similar manner. If this technology is mature enough, people are likely to be very free to manipulate genetic material. Although the experimental procedures used in recombinant DNA technology, the enzymes used to construct recombinant DNA, and various reagents are not new, the innovation of this technology is the reorganization of DNA, cutting and reconnecting the DNA.

In the winter of 1970, Berg and his postdoctoral fellow, David Jackson, began their first attempt: cutting and joining two DNA fragments. Their experimentation was very tedious at the time and Berg described the experiment as a nightmare for biochemists. They first need to purify the DNA, mix the DNA with the enzyme, and then perform purification on a cold column, and they need to continually repeat the above steps to optimize the biochemical reaction conditions. The problem is that the shearing enzymes they use are not very efficient and the yield is very low.

Despite technical limitations, Berg and Jackson eventually succeeded in linking the entire genome of SV40 to one DNA fragment of lambda phage and three genes derived from E. coli. Both SV40 and lambda phage are viruses, but there is a huge difference between them. E. coli is a completely different organism than SV40. Berg and Jackson successfully combined these DNAs from different organisms. Berg decided to call this hybrid DNA a recombinant DNA.

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