What is the secret of the "1.2-million-dollar anticancer drug"?

Recently, the news of "$1.2 million anti-cancer drug" has pushed CAR-T therapy to the people. Most people are more curious and have questions about this new therapy than about its expensive price.

The principle of CAR-T therapy is not difficult to understand. Simply, it is the modification of T-lymphocytes in the patient's body and the installation of a "navigation device" to precisely strike cancer cells. This technology involves a number of molecular biology techniques, such as size-selection, transduction, amplification and so on. Today, the article is going to talk about amplification.

The term amplification refers to the polymerase chain reaction (PCR), a molecular biology technique used to amplify specific DNA fragments, which can be thought of as a special DNA replication outside of the organism. When a high copy number is amplified by using the PCR reaction, the next step of how to detect it becomes critical.

Traditional gel electrophoresis PCR and second-generation fluorescence quantitative PCR rely on standard curves or reference genes to determine the number of nucleic acids, and can only achieve relative quantification. Digital PCR (dPCR) was first proposed by Bert Vogelstein et al. in PNAS, the Proceedings of the National Academy of Sciences, at the end of the 20th century. This new method for absolute quantification of nucleic acid molecules based on the Poisson distribution principle, which has the advantages of high sensitivity, high precision, and high resistance to interference, is known as the third generation PCR technology.

The basic principle of digital PCR is to partition the reaction system. A fluorescent PCR reaction system containing tens of microliters of nucleic acid molecules is randomly partitioned into tens of thousands of tiny independent reaction systems.

The process of template DNA molecules entering each small reaction system satisfies the Poisson distribution principle, and the intensity of the fluorescence signal of the amplified PCR product is detected. The number of DNA molecules is counted directly. Based on the relative ratio and the volume of the reactor, the nucleic acid concentration of the starting template DNA molecules can be deduced from the Poisson distribution, and its absolute quantification.

Digital PCR can be divided into three main types of systems, namely microplate, microchamber and microdrop, according to the different forms of their reaction units. At present, two forms of microdrop type and chip type are mainly used in the market.

As the third generation PCR technology, the advantages of digital PCR are mainly as follows.

▶ High sensitivity and absolute quantification at single-molecule level: A traditional PCR reaction is turned into tens of thousands of PCR reactions, and the target sequences are detected independently in these tens of thousands of reaction units, independent of standard curves and amplification kinetics, without relying on Ct values, which improves the sensitivity of detection.

▶ High accuracy and reproducibility for detecting small differences: dPCR keeps samples completely isolated from each other, which can effectively prevent sample cross-contamination and reduce the pipetting process. dPCR can detect low-abundance target sequences from background sequences and has high sensitivity, making it particularly suitable for detecting rare mutations in complex backgrounds.

▶ Strongly tolerated inhibitors for a variety of complex samples: Blood, stool, food, soil and other samples contain a large number of inhibitors for PCR reactions. Since digital PCR distributes the target sequence into multiple independent reaction systems, it significantly reduces the interference of background signals and inhibitors to the reaction. The amplification matrix effect is greatly reduced, making it suitable for accurate quantitative detection of gene expression in complex samples.

Digital PCR technology provides new ideas for diverse nucleic acid detection needs in different scenarios, especially in medical testing, showing good application prospects in early detection of infectious diseases, liquid biopsy of cancer, and non-invasive prenatal testing.

With the increase in demand for precision diagnosis and treatment, the domestic and international markets for digital PCR are relatively active. Major foreign companies are actively laying out digital PCR business. The domestic digital PCR industry started late and most companies are still in the early planning and development stage, but in the face of its unparalleled advantages, digital PCR has become one of the hot areas that have received wide attention in recent years. It is believed that as new molecular biology technologies such as CAR-T therapy enter the sight of the general public, technologies such as digital PCR will certainly usher in a vigorous development.