From PCR to qPCR: A brief history of the evolution of theory and application

Since PCR entered the molecular biology arena as a patented biotechnology in 1985, no other biotechnology has had such a profound impact on the development of the entire life sciences, with so many cited papers and such a wide range of applications. PCR is widely used in life sciences, medical diagnosis, forensic testing, food hygiene and environmental testing due to its high sensitivity and high efficiency in amplifying target DNA.

PCR ( P olymerase C hain R eaction), translated into Chinese as "polymerase chain reaction", simply put, under the action of two short nucleotides (primers) and heat-resistant DNA polymerase, the DNA template to be amplified is first heated to denature and melt, and then cooled to a certain temperature, the primer binds to the single strand of the DNA to be amplified, and then the temperature is raised to allow the annealed primer to extend under the action of DNA polymerase. This denaturation-annealing-extension process is a PCR cycle, which is repeated continuously, and the DNA template can be doubled in just tens of minutes. ⁿ The whole reaction process is as follows:

The basic reagents required for a routine PCR reaction include:

• DNA template contains the DNA fragment that needs to be amplified.
• A pair of primers determines the starting and ending positions of the amplification.
• DNA polymerase copies the area to be amplified.
• Deoxynucleoside triphosphates (dNTPs) are used to construct new complementary chains.
• The buffer system containing magnesium ions provides a chemical environment suitable for the polymerase to function.

In current mature PCR reagents, reaction reagents other than primers and templates are often configured into a Mix according to the parameters of the optimized reaction. During the experiment, the target fragment and related primers are directly added to the finished Mix, which greatly simplifies the reagent addition process in the PCR experiment.

So how did such an epoch-making technology in the field of molecular biology come into being? Let us expand this history a little bit. Let us go back to the timeline 1953 Year What happened that year? The first five-year plan of the new China? Audrey Hepburn won the Oscar for Best Actress for "Roman Holiday"? Uh uh, I'm getting off topic... (ノ｀Д´)ノ Of course, Watson and Crick proposed that DNA is a long chain of double-helix deoxynucleotides that are parallel in opposite directions through complementary pairing of bases! This is the historical photo that is widely circulated in the biological community:

The person sitting on the left in the picture above and looking up is Watson, and the person standing on the right and pointing at the model with high spirits is Crick^_^ By the way, Crick died of illness in 2004, while Watson is still alive, but Crick was originally twelve years older than Watson...

Based on this structure, they further speculated that this might imply the replication mechanism of genetic material, but this needed to be proved by experiments. In fact, around the time when the DNA double helix structure model was proposed, the team led by Kornberg was studying the mechanism of DNA replication. In 1956, he confirmed that DNA is a molecule that can replicate itself, and in 1957, he identified the first DNA polymerase. Although the function of this enzyme is very limited, it opened the door to the study of the DNA replication mechanism. In 1959, Kornberg was awarded the Nobel Prize in Physiology or Medicine for discovering the bacterial DNA replication mechanism and reproducing the DNA replication process in a test tube. In 1962, Watson and Crick also won the Nobel Prize in Physiology or Medicine for the DNA double helix structure model.

In 1969, Thomas Brock, a microbiologist at Indiana University, and his graduate student Hudson Freeze discovered a thermophilic Thermotoga marine fungus from the volcanic hot springs in Yellowstone National Forest. T.aquaticus (Taq), which laid a solid foundation for Trela and his Chinese graduate student Alice Chien to separate and purify the heat-stable Taq DNA polymerase that can tolerate >75°C in 1976 (Deoxyribonucleic Acid Polymerase from the Extreme Thermophile Thermus aquaticus). Gobind Khorana, an Indian scholar who won the Nobel Prize in 1968 for discovering the genetic code and its function in protein synthesis, and his postdoctoral fellow Kleppe and others first published an article in the Journal of Molecular Biology in 1971 that was later called "Guiding PCR to the Feasibility of Technology". He wrote in this article:

"If a double-stranded molecule wants to have two identical structures, each containing the entire length of the template chain including the primer, it needs to add DNA The polymerase completes such a Patching replication The process ends with one original double-stranded molecule producing two identical double-stranded molecules. The entire cycle needs to be repeated continuously, and fresh polymerase must be added each time.

In this article, the young man Kleppe from Norway clearly proposed the concept of repair replication, which was later called the prototype of PCR technology by the academic community. However, at that time, sequencing technology had not been invented, and thermostable DNA polymerase had not been discovered. In addition, synthetic primers were still just a kind of scientific performance art, so this idea was quickly forgotten by the academic community. However, good ideas are like a bunch of sparks, and scattered sparks always accidentally ignite a fire. In fact, the work of this article was as early as the Gordon Conference held in New Hampshire in 1969. Kleppe described to the participants the technology of producing two double-stranded molecules of the same structure from a double-stranded DNA molecule, and the Gordon Conference is like the annual "Huashan Sword Contest" of the American academic community. It is worth mentioning that among the audience at the time was a professor named Stuart Linn, who demonstrated the experiment in his subsequent teaching using the reaction components described by Kleppe. Among the students attending the lecture was Karry Mullis, who took over the baton of PCR research and completed the final sprint.

In 1979, Sanger published an article titled "Chain-terminated DNA sequencing" in the Proceedings of the National Academy of Sciences (PNAS). In this article, Sanger mentioned that oligonucleotide primers, DNA polymerase and modified nucleic acids that can terminate primer extension reactions can be used in DNA sequencing experiments. Just one year later, in 1980, Sanger won the Nobel Prize again for inventing the Sanger nucleic acid sequencing method. The subsequent improved automated Sanger sequencing method achieved the "Human Genome Project" jointly carried out by many countries. By 1980, almost the entire academic community knew that DNA sequencing and reverse transcription of cDNA for cloning and expression could be performed through DNA polymerase primer extension. More importantly, the nick translation of DNA polymerase was used for Southern blotting probe labeling. This DNA hybridization test can be used for semi-quantitative detection of target DNA fragments. Everything nurtured and hinted that the PCR technology method was about to come into the world, just waiting for one person to appear, he was Dr. Karry Mullis.

Due to space limitations, we will only show you the historical moments when PCR inspiration flashed. After all, no matter how much history there is, people will get bored sometimes...٩(๑´0`๑)۶ Of course, the plot foreshadowing before the historical moment is still indispensable. In 1972, 28-year-old Karry Mullis received a Ph.D. in biochemistry from the University of California, Berkeley. However, in his six years of doctoral research, he did not actually publish a paper related to biochemistry. Instead, he published a paper in Nature magazine in 1968 titled "The Cosmological Significance of Time Reversal" to help Mullis get his PhD...(;￢д￢)

In 1979, Mullis finally joined a private biotechnology company called Cetus in the San Francisco Bay Area. At that time, biotechnology companies were still in their infancy, and few academics were willing to leave the ivory tower to work for private companies, because this was usually considered the end of one's academic career (just like the difficulty of finding a job for a biotech dog today). However, Cetus was a very special company. This company brought together a group of capable and dreamy scientists, who worked together towards their goals in a free and open atmosphere, which was very different from the practice of professors and laboratory directors in general colleges at that time who closed their doors and did their own thing. Cetus hired Mullis to rely on his expertise in organic chemical synthesis to synthesize oligonucleotide DNA molecules less than 20nt in length for research and development by other departments within the company. Mullis later recalled that because the company had already achieved automated operations, his department produced so many nucleotide fragments that there was no space in the refrigerator to store them, and the department staff were leisurely and had nothing to do.

Because he was not busy at work, Mullis began to have time to ponder the denaturation and renaturation characteristics of the nucleotide fragments he produced. Through continuous experiments, he also figured out a quantitative calculation formula. He deduced that if it could grow exponentially, it would become a powerful tool for quickly obtaining a large number of DNA fragments. Coincidentally, Mullis had been fascinated by how computers handle logarithmic functions at the time, which led him to connect two things together. Every molecular biologist knows about the DNA replication reaction (doubling) and knows about exponential functions. But only Mullis thought of two things at the same time, which gave rise to the idea of exponentially amplifying DNA. Mullis realized that if small things continue to double, they will increase very quickly. For example, doubling a particular DNA fragment 30 times will produce 2 ³⁰ Copies of DNA, so many copies of DNA are enough for researchers to conduct relevant scientific research. This idea finally broke out one day. According to his own account, it was a Friday night in May 1983. Mullis drove a silver Honda Civic and took his colleague and his Nth girlfriend Jennifer (yes, one of his hobbies is to pick up girls, and he has been married four times so far) from the Bay Area of California to a country house in Mendocino County to spend a pleasant and romantic weekend. Driving on the winding 128 highway in Northern California, the winding and tortuous road gave him inspiration. He had such a picture in his mind: the winding mountain road is a double strand of DNA, the car driving on the two-lane road is the primer for amplification, and the exhaust gas emitted by the car tail pipe is like the synthesized complementary base sequence. So he stopped the car, woke up his sleeping girlfriend, and excitedly explained his idea.

Mullis originally thought that such a simple idea should have been proposed by someone, but after searching the literature, he found that no one had. In the 3 to 5 months after the "sudden epiphany", Mullis did not take any action. The reason for this is unknown, but according to insiders, it either means that the busy work pace of Cetus has left Mullis no time to take care of it, or that Mullis is caught up in an endless love affair, or another possibility is the suspicion and lack of support from colleagues. In August of the same year, Mullis made his first formal report on the principle of PCR in the company, and the audience responded coldly. First, everyone has become accustomed to his wild thoughts; second, most people think that this principle is too simple. If it is feasible, someone must have done it long ago. Otherwise, there must be something that is not feasible, but no one can clearly say why it is not feasible.

So Mullis had to start proving the feasibility of this idea. Starting from September 1983, Mullis conducted a series of experiments, changing several DNA templates and trying different heating and cooling cycles, but the results were not ideal. At most, he only found a faint band on the electrophoresis gel, which could not convince others that PCR had the effect of amplification.

The energy and time required for any research method from the concept to the practical application stage are mostly underestimated by the general public. Since Mullis had not received any training in molecular biology before, the company sent technicians to assist him, a total of three. These people played an important role in the development of PCR. In November 1984, Mullis's technicians obtained credible results for the first time, proving the feasibility of PCR. So in early 1985, the company decided to send Randall Saiki, a skilled Japanese technician, again. This was a correct decision. Before the emergence of automated instruments, PCR was a very cumbersome technology, requiring many people to keep busy in a pile of test tubes, stopwatches, and water baths of different temperatures, and even independent working spaces to prevent possible contamination. This required long-term repeated operations, and people who were not agile could not do it. Saiki's results were clean and beautiful, which was convincing.

During the PCR operation, repeated heating and cooling cycles are required. The E. coli DNA polymerase used in the previous cycle denatures and becomes inactivated at high temperatures. Therefore, new polymerase must be added after each cycle. This approach is not only cumbersome, but also expensive. At the time, the polymerase required for one cycle was worth $1, and 30 cycles would cost $30, not to mention more cycles. Therefore, in the spring of 1986, Mullis first proposed the idea of using a high-temperature resistant enzyme. After a literature search, two relevant papers were found. The earlier one was done in the United States, and the other was the result of Russian scientists.

The first report on the isolation of a thermostable DNA polymerase was by a young Taiwanese scientist named Qian Jiayun. In 1973, Qian Jiayun went to study biology at the University of Cincinnati in Ohio. Her mentor, J. Trela, studied a thermophilic bacterium found in a hot spring in Yellowstone Park. Thermus aquaticus Qian Jiayun was curious about the bacteria and asked another American student to use the bacteria as the topic of their paper research. Under the guidance of another teacher, Qian Jiayun successfully isolated the thermostable Taq DNA polymerase from the bacteria and published the research results in 1976. Journal of Bacteriology superior.

Although Mullis proposed to apply Taq DNA polymerase to PCR, there was no ready-made enzyme available at that time, so he had to find a way to separate it himself. Cetus had a full set of equipment for separating proteins, and there were people willing to guide him, but Mullis was a procrastinator. After waiting for a few months, the rest of the company had to do it themselves. Following the steps previously published by Qian Jiayun and others, they purified Taq DNA polymerase in three weeks. In June 1986, Saiki applied it to PCR for the first time, and the effect was amazing. It can be said that it was a hit. Taq DNA polymerase not only greatly simplified the PCR work, but also had stronger specificity and activity than the previously used Escherichia coli DNA enzyme, and the background bands were almost eliminated. Since then, PCR has been a great success. In December 1991, Hoffmann Roche Pharmaceuticals reportedly purchased Cetus's PCR technology patent for $300 million, and Cetus company also became history. Until recent years, due to the work previously published by Qian Jiayun and others, the patent rights of Taq DNA polymerase were challenged, and the patent of PCR was also affected, but that is another story.

PCR can efficiently amplify trace amounts of DNA molecules in vitro through only three steps: "denaturation", "annealing" and "extension", which greatly facilitates the development of molecular biology. Bio-Raid has even written a song called "PCR Song" to praise this milestone technological innovation:

Well, the image is quite strong, with the following lyrics. PCR Perfect presentation of past and present life ^_^

There was a time when to amplify DNA,

You had to grow tons and tons of tiny cells.

Then along came a guy named Dr. Kary Mullis,

Said you can amplify in vitro just as well.

Just mix your template with a buffer and some primers,

Nucleotides and polymerases, too.

Denaturing, annealing, and extending.

Well it's amazing what heating and cooling and heating will do.

PCR, when you need to detect mutations.

PCR, when you need to recombine.

PCR, when you need to find out who the daddy is.

PCR, when you need to solve a crime

The singing comes to an end first, and now let's get down to business╭(￣▽￣)╯… The driving force behind the continuous updating of PCR technology is that the DNA polymerase that synthesizes new chains based on templates has been constantly developing. In today's diverse PCR applications, it is no longer possible to rely on a single wild-type Taq enzyme to go everywhere. Kapa Biosystems has begun to adopt the concept of high-throughput "directed evolution" technology to design specialized DNA polymerases for special PCR applications, such as NGS. "We screen a large number of special proteins to find mutants that greatly enhance enzyme function," said John Foskett, technical director of Kapa Biosystems, who designed the high-fidelity KAPA HiFi DNA polymerase specifically for NGS library amplification. "This product has been optimized several times to effectively reduce the preference during PCR amplification and increase the yield, thereby obtaining more consistent sequencing coverage and increasing the diversity of the library."

With the continuous development of biological experiment needs, PCR In the course of its development, technology has gradually evolved into a series of focus on different experimental purposes and applications. PCR Classification, the more common ones include: Touchdown PCR , Multiplex PCR , qPCR by ddPCR Here, we focus on qPCR The basic principles and their wide applications.

Quantitative PCR, also known as real-time PCR, is a method that uses fluorescent dyes or fluorescently labeled probes to detect the total amount of products after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction, and is recognized by the optical detection system in the qPCR instrument in the form of a fluorescent signal. Finally, a quantitative analysis of unknown sample templates is performed by drawing a relevant standard curve. Compared with traditional PCR, qPCR can perform quantitative analysis on samples.

After conventional PCR, the PCR products obtained will be subjected to agarose gel electrophoresis, which is a simple qualitative analysis.

qPCR introduces fluorescent groups (dyes or probes) into the reaction system, which can be used to label and track PCR products and monitor the reaction process online in real time. Combined with appropriate software, the products can be analyzed and the initial concentration of the sample template to be tested can be calculated. This is a very accurate quantitative method.

Since the starting template needs to be accurately quantified, there are two specific parameters in the qPCR results that are critical to the evaluation process. The first parameter is the amplification curve. Taking the following figure as an example, the horizontal axis represents the number of cycles, and the vertical axis represents the fluorescence intensity or relative fluorescence intensity. At the beginning of the reaction, the fluorescence signal is unstable and fluctuates. Then the signal tends to stabilize and grows exponentially. After reaching a certain number of cycles, the fluorescence signal intensity no longer increases and remains stable. The amplification curve is presented as an S-shaped curve, including: baseline period, exponential amplification period, and plateau period. After the reaction is completed, the qPCR instrument will generate a threshold line with 10 times the standard deviation of the baseline fluorescence signal. The threshold line and the amplification curve will intersect. The horizontal axis corresponding to the intersection represents the Ct value. The meaning of the Ct value represents the number of amplification cycles experienced when the fluorescence signal intensity reaches the threshold in each reaction system, which is the basis for subsequent quantitative calculations.

The second important parameter is the melting curve. The melting curve is detected after the reaction is completed, reflecting the relationship between temperature and fluorescence value. This type of detection is only applicable to the dye method. The probe method cannot be analyzed because the probe cannot be reduced after hydrolysis. From the results of the derivation in the figure below, it can be seen that one peak corresponds to a drop in the fluorescence signal. Each drop represents a large amount of denaturation of a double-stranded product within this temperature range. Therefore, a single peak represents only one specific product, and more than one peak represents the presence of non-specific amplification products or primer dimers. The melting curve helps us determine the specificity of the reaction. Through the analysis of amplification curves and melting curves, only complete and specific reactions can give authentic Ct values, which can be used for subsequent quantitative calculations.

For subsequent data analysis of qPCR experimental results, the following linear function is usually used:

Define the initial template amount as X0, and the product amount after the nth cycle as X _n , then under ideal PCR conditions, X _n =X₀ ×2n, under non-ideal PCR conditions, we define the primer amplification efficiency as Ex, Xn=X₀ ×(1+Ex)n, take the logarithm of both sides, substitute the Ct value and the amount of product when reaching Ct X(Ct) into the formula, lg X₀ = (- lg(1+Ex) )×C(t)+ lg Xc(t)This final equation shows that the logarithm of the initial template concentration is linearly related to the Ct value. Based on this linear relationship, the Ct value can be used to calculate the subsequent expression level.

In real scientific research, we often need to consider more complex parameters than amplification curves and melting curves. review , The MIQE Guidelines , which provides the minimum information standards required for publishing an article. qPCR terminology, concepts, research and clinical applications, sample collection, processing and preparation, nucleic acid quality control, reverse transcription, qPCR The operational standards and specifications for processes and data analysis are elaborated in detail.

Although there are many brands and models of qPCR instruments, from the perspective of working principles, they all include the following three reaction modules: excitation light emission source, receiving device, and PCR reaction module.

Since fluorescent groups are introduced into the system, the excitation light source needs to emit light of a certain wavelength, which will reflect light of another wavelength when encountering the fluorescent group, and then be received by the receiving device in real time. It is precisely because the qPCR instrument has these two modules added compared to the ordinary PCR instrument that the consumables of qPCR have higher requirements than those of ordinary PCR, and the light transmittance of its top cover must be good. When doing qPCR, you cannot touch the top cover with your bare hands or wearing latex gloves. You must wear PE gloves to prevent impurities from remaining on the top cover and affecting the emission and reception of fluorescent signals.

According to the quantitative method of qPCR, it can be divided into SYBR dye method, TaqMan probe method and molecular beacon.

The SYBR dye method uses the characteristic that SYBR Green I molecules can bind to dyes with green excitation wavelengths in the minor groove region of the double helix of all double-stranded DNA (dsDNA) to achieve quantification. SYBR Green I only fluoresces after binding to double-stranded DNA, and free dye molecules do not emit light. During the extension of the newly synthesized chain, SYBR Green I is incorporated into the double helix. During denaturation, the DNA double helix is unwound, and SYBR Green I is freed and has no fluorescence. Since non-specific amplification products and primer dimers are both dsDNA, the SYBR dye method can only ensure specificity through primers. Its advantages are simplicity and low cost, and it is suitable for scientific research customers with small sample quantities.

TaqMan The core of the probe method is the probe molecule. TaqMan The probe is single-stranded DNA , 5 'End coupled luminescent group, 3 'The quenching group is coupled to the end, and no fluorescence signal can be detected for the free intact probe. The fluorescence emitted by the luminescent group will be absorbed and quenched by the quenching group. The probe is hydrolyzed, and the fluorescence signal can be detected when the luminescent group and the quenching group are separated.

At the beginning of the qPCR reaction, the double-stranded template is heated and denatured to melt into a single strand. The TaqMan probe anneals with the template strand first, and the primer then anneals to the template, followed by single-strand extension. During the extension process, the Taq enzyme exerts 5'-3' exonuclease activity, and when it encounters the probe, it will remove the probe base by base from the 5' end. The luminescent group will separate from the quenching group, so the fluorescence detection system can receive the fluorescent signal. Each time a DNA strand is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescent signal and the formation of the PCR product are synchronized. The specificity of the TaqMan probe method is not only provided by the primer, but also guaranteed by the probe molecule. Because of its higher annealing temperature, the TaqMan probe method has better specificity. By adding multiple probes to a reaction system, multiple genes can be detected simultaneously.

Molecular beacons are similar to TaqMan probes. In the free state, the probes complement each other to form a hairpin structure, with the 5' end coupled to the luminescent group and the 3' end coupled to the quenching group. In this state, the luminescent group and the quenching group are close to each other, and the fluorescence emitted by the luminescent group will produce fluorescence resonance energy transfer (FRET). After the quenching group is excited, the signal decays. When the temperature of the reaction system rises, the probe with the hairpin structure is opened, and the stem-loop region of the molecular beacon anneals and combines with the template chain. After the luminescent group and the quenching group are separated, the FRET phenomenon will not occur due to the long distance. The released fluorescence signal is detected by the receiving device in the machine, and the newly synthesized complementary chain replaces the molecular beacon. The beacon molecule that has separated from the template chain re-forms the hairpin structure and no longer releases the fluorescence signal.

To summarize the choice of quantitative method, in general, in scientific research, most quantitative methods will choose cheap and convenient SYBR Dye method, if there is a more stringent quantitative requirement, you can choose TaqMan Probe method; in medical examination, accurate and specific TaqMan Probe method. In addition, SYBR The dye method is suitable for reactions where the specificity requirement is not particularly high and the number of molecules (copy number) exceeds 1000 reaction, pre-experiment before probe experiment, PCR The conditions are very mature, there are no dimers, and there is no non-specific amplification; TaqMan The probe method is suitable for experiments with high specificity requirements and multiple PCR (labeled with different fluorescent groups), SNP Experiments requiring high detection and sensitivity. Molecular beacons have extremely high background fluorescence.