From PCR to qPCR: A Brief History of the Evolution of Theory and Application

Since PCR, a patented biotechnology, entered the molecular biology arena in 1985, no other biotechnology has had such a profound impact on the development of life sciences, with such numerous citations and widespread applications. PCR, with its high sensitivity and high efficiency in amplifying target DNA, is widely used in life sciences, medical diagnostics, forensic testing, food hygiene, and environmental testing.

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, then cooled to a certain temperature, the primers bind to the single strand of the DNA to be amplified, and then the temperature is raised to allow the annealed primers to be extended under the action of DNA polymerase. This denaturation-annealing-extension process is a PCR cycle, which is repeated continuously and can perform 2-fold DNA amplification on the DNA template in just tens of minutes. ⁿ The entire reaction process is as follows:

The basic reagents required for a conventional PCR reaction include:

• DNA template, which contains the DNA fragment to be amplified.
• A pair of primers determines the starting and ending positions of the amplification.
• DNA polymerase copies the region 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 today's mature PCR reagents, reaction reagents other than primers and templates are often configured into a mix according to optimized reaction parameters. During the experiment, the target fragment and related primers are directly added to the finished mix, greatly simplifying 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 So, what happened that year? New China's first Five-Year Plan? Did Audrey Hepburn win the Best Actress Oscar for "Roman Holiday"? Uh huh, I'm getting off track... (ノ｀Д´)ノ Of course, Watson and Crick proposed that DNA is a long, parallel double-helix of deoxynucleotides formed by complementary pairing of bases! And that's what this historical photo, now widely circulated in biology circles, is about:

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

Based on this structure, they further speculated that it might hint at a mechanism for the replication of genetic material, though this required experimental confirmation. In fact, around the same time that the DNA double helix model was proposed, a team led by Kornberg was studying the mechanism of DNA replication. In 1956, he confirmed that DNA is a self-replicating molecule, and in 1957, he identified the first DNA polymerase. Although this enzyme had limited functions, it opened the door to further research into the mechanism of DNA replication. In 1959, Kornberg was awarded the Nobel Prize in Physiology or Medicine for his discovery of the bacterial DNA replication mechanism and for reproducing the DNA replication process in a test tube. In 1962, Watson and Crick also received the Nobel Prize in Physiology or Medicine for their work on the DNA double helix model.

In 1969, Thomas Brock, a microbiologist at Indiana University, and his graduate student Hudson Freeze discovered a thermophilic Thermotoga marine fungus from a volcanic hot spring in Yellowstone National Forest. T.aquaticus This laid a solid foundation for Trela and his Chinese graduate student Alice Chien's subsequent isolation and purification of the thermostable Taq DNA polymerase (Deoxyribonucleic Acid Polymerase from the Extreme Thermophile Thermus aquaticus), which tolerates temperatures exceeding 75°C, in 1976. Gobind Khorana, an Indian scholar who won the 1968 Nobel Prize for his discovery of the genetic code and its function in protein synthesis, and his postdoctoral fellow, Kleppe, and others, published the first article in the Journal of Molecular Biology in 1971, which later became known as "The Guide to the Technical Feasibility of PCR." In this article, Kleppe wrote:

“To obtain two identical structures of a double-stranded molecule, each containing the entire base length of the template chain including the primer, one needs to add DNA The polymerase completes such a Patch Replication The process ends with the production of two identical double-stranded molecules from one original double-stranded molecule. The entire cycle needs to be repeated continuously, and fresh polymerase must be added each time."

In this article, the young Norwegian Kleppe clearly proposed the concept of repair replication, which later became known as PCR. However, at the time, sequencing technology had not yet been invented, thermostable DNA polymerase had not yet been discovered, and primer synthesis was still a scientific art form, so the idea was quickly forgotten. However, a good idea is like a spark, and scattered sparks often accidentally ignite a fire. In fact, the work of this article was already described at the Gordon Conference in New Hampshire in 1969. Kleppe described the technique of generating two identical double-stranded molecules from a single double-stranded DNA molecule to attendees. The Gordon Conference is like the annual "Huashan Sword Contest" of American academia. It is worth mentioning that among the audience at the time was Professor Stuart Linn, who subsequently demonstrated the experiment in his teaching using the reaction components described by Kleppe. Among the students present 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 described the use of oligonucleotide primers, DNA polymerase, and modified nucleic acids capable of terminating primer extension reactions for DNA sequencing experiments. Just one year later, in 1980, Sanger was awarded the Nobel Prize again for inventing the Sanger nucleic acid sequencing method. Subsequent improvements to the automated Sanger sequencing method enabled the multinational Human Genome Project. By 1980, nearly the entire academic community was aware that DNA polymerase primer extension could be used for DNA sequencing and reverse transcription of cDNA for cloning and expression. More importantly, DNA polymerase nick translation was used for probe labeling in Southern blotting, and this DNA hybridization assay could be used for semi-quantitative detection of target DNA fragments. All of this was foreshadowing the imminent arrival of PCR technology, waiting for the arrival of one person: Dr. Karry Mullis.

Due to space limitations, we'll only highlight the historic moments when the inspiration for PCR emerged. After all, even with so much history, one can get bored eventually... Of course, the plotting before these historic moments is essential. In 1972, 28-year-old Karry Mullis received his Ph.D. in biochemistry from the University of California, Berkeley. However, during his six years of doctoral research, he didn't actually publish a single biochemistry-related paper. Instead, it was his 1968 Nature paper, "The Cosmological Significance of Time Reversal," that secured Mullis his PhD... (;￢д￢)

In 1979, Mullis finally landed a job at Cetus, a private biotech company in the San Francisco Bay Area. At the time, biotech companies were still in their infancy, and few academics were willing to leave the comforts of their academic world for private industry, often seen as the end of their academic careers (much like the difficulty finding a job in biology today). However, Cetus was a unique company, bringing together talented and ambitious scientists who worked together towards a common goal in a free and open environment. This was a stark contrast to the closed-door approach of professors and laboratory directors at the time. Cetus hired Mullis to leverage his expertise in organic chemical synthesis, tasked with synthesizing oligonucleotide DNA molecules less than 20 nucleotides in length for use by other departments within the company. Mullis later recalled that, due to the company's advanced automation, his department produced so many nucleotide fragments that the refrigerators ran out of room, leaving the staff idle and unemployed.

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 worked 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 the exponential function. But only Mullis thought of two things at the same time, which led to the idea of exponential amplification of DNA. Mullis realized that if small things continue to double, they will increase very quickly. For example, doubling a specific DNA fragment 30 times will produce 2 ³⁰ Copies of DNA—enough copies for researchers to conduct relevant scientific research—finally struck him. According to his own account, it was a Friday night in May 1983. Mullis was driving his silver Honda Civic, accompanied by his colleague and then-girlfriend, Jennifer (yes, one of his hobbies was picking up girls, and he'd been married four times), from the Bay Area to his cabin in Mendocino County for a pleasant and romantic weekend. Driving along Northern California's winding Highway 128, the twists and turns of the road gave him a sudden inspiration. He imagined the winding mountain road as a double strand of DNA, the cars on the two-lane road as primers for amplification, and the exhaust fumes from their tailpipes as the synthesized complementary base sequences. He stopped the car, woke his sleeping girlfriend, and excitedly explained his idea.

Mullis initially assumed such a simple idea must have been proposed before, but a literature search revealed none. For three to five months after his "eureka moment," Mullis took no action. The reasons for this are unknown, but those familiar with the matter recall that it could have been due to the frenetic pace of work at Cetus, his inability to focus on the topic, his ongoing romantic relationship, or perhaps even the skepticism and lack of support from his colleagues. In August of that year, Mullis gave his first formal presentation on the principles of PCR at the company, to a lukewarm reception. For one thing, everyone was accustomed to his wild speculations; for another, most people assumed the principle was too simple. If it worked, it must have been done before, or else, there must be some reason it wasn't feasible. But no one could clearly articulate why.

Mullis set out to prove the feasibility of his idea. Starting in September 1983, he conducted a series of experiments, switching DNA templates and experimenting with different heating and cooling cycles. The results were unsatisfactory; at best, he could only find a faint band on the electrophoresis gel, which was unsatisfactory in convincing anyone that PCR was working.

The effort and time required to develop any research method from conception to practical application are often underestimated. Since Mullis had no prior training in molecular biology, the company sent three technicians to assist him. These individuals played a crucial role in the development of PCR. In November 1984, Mullis's technicians achieved their first reliable results, proving the feasibility of PCR. So, in early 1985, the company decided to bring on the highly skilled Japanese-American technician, Randall Saiki. This proved to be a wise decision. Before the advent of automated instrumentation, PCR was a tedious process, requiring numerous people to maneuver through a multitude of test tubes, stopwatches, and water baths at varying temperatures, even requiring dedicated workspaces to prevent contamination. This involved lengthy, repetitive manipulations, making it impossible for anyone without nimble hands. Saiki's results were clean, beautiful, and convincing.

The PCR process requires repeated cycles of heating and cooling. However, the E. coli DNA polymerase used in the previous cycle denatures and becomes inactivated at high temperatures, so fresh 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 cost $1, which translates to $30 for 30 cycles, not to mention more cycles. Therefore, in the spring of 1986, Mullis first proposed the idea of using a thermostable enzyme. After a literature search, he found two relevant papers: the earlier one was conducted in the United States, and the other was the work of Russian scientists.

The first report on the isolation of a thermostable DNA polymerase was by a young Taiwanese scientist, Chia-Yun Chia-Yun. In 1973, Chia-Yun went to the Department of Biology at the University of Cincinnati in Ohio. Her advisor, J. Trela, studied a thermophilic bacterium found in a hot spring in Yellowstone National Park. Thermus aquaticus Under the guidance of another teacher, Qian Jiayun successfully isolated the high-temperature resistant Taq DNA polymerase from the bacteria and published the research results in 1976. Journal of Bacteriology superior.

Although Mullis proposed applying Taq DNA polymerase to PCR, the enzyme wasn't readily available at the time, so he had to find a way to isolate it himself. Cetus had a full suite of protein separation equipment and someone willing to provide guidance, but Mullis was a chronic procrastinator. After waiting for several months, the rest of the company had to take matters into their own hands. Following the procedures previously published by Qian Jiayun and others, they purified Taq DNA polymerase in just three weeks. In June 1986, Saiki first applied it to PCR, with astonishing results—an instant success. Taq DNA polymerase not only greatly simplified PCR work but also boasted greater specificity and activity than the previously used E. coli DNA enzyme, virtually eliminating background bands. From that point on, PCR became a resounding success. In December 1991, Hoffmann-Roche Pharmaceuticals purchased Cetus's PCR technology patents for a reported $300 million, and Cetus itself faded into 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 achieves efficient amplification of trace amounts of DNA molecules in vitro through just three steps: denaturation, annealing, and extension. This has greatly facilitated the development of molecular biology, so much so that Bio-Raid has written a song called "PCR Song" to celebrate this milestone technological innovation:

Well, the visual effect is quite strong, with the following lyrics. PCR The past and present life is perfectly presented ^_^

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

Let's stop singing for now, and 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 be universal. 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 screened 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 bias during PCR amplification and increase yield, resulting in more consistent sequencing coverage and increased library diversity."

With the continuous development of biological experiment needs, PCR In the course of its development, technology has gradually evolved into a series of PCR Classification, the more common ones include: touchdown PCR 、 multiplex PCR 、 qPCR by ddPCR Here, we focus on qPCR The basic principles and its wide application.

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 product after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction. This fluorescent signal is recognized by the optical detection system in the qPCR instrument, and then a standard curve is drawn to quantify the unknown sample template. Compared to traditional PCR, qPCR can be performed 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, monitor the reaction process in real time online, and analyze the products in combination with corresponding software to calculate the initial concentration of the sample template to be tested. It is a very accurate quantitative method.

Because the starting template needs to be accurately quantified, two specific parameters in the qPCR results are crucial to the evaluation process. The first is the amplification curve. Taking the figure below as an example, the horizontal axis represents the cycle number, 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. The signal then stabilizes and grows exponentially. After reaching a certain number of cycles, the fluorescence signal intensity no longer increases and remains stable. The amplification curve is displayed as an S-shaped curve, including: a baseline phase, an exponential amplification phase, and a plateau phase. After the reaction is completed, the qPCR instrument generates a threshold line at 10 times the standard deviation of the baseline fluorescence signal. The threshold line intersects the amplification curve, and the horizontal axis corresponding to the intersection represents the Ct value. The Ct value represents the number of amplification cycles experienced when the fluorescence signal intensity in each reaction system reaches the threshold value and forms the basis for subsequent quantitative calculations.

The second important parameter is the melting curve. This is measured after the reaction is complete and reflects the relationship between temperature and fluorescence. This type of analysis is only applicable to dye-based methods; probe-based methods cannot perform this analysis because the probe cannot be reduced after hydrolysis. As can be seen from the derivative results in the figure below, each peak corresponds to a drop in fluorescence signal. Each drop represents a significant melting of a double-stranded product within this temperature range. Therefore, a single peak indicates a single specific product, while more than one peak indicates the presence of nonspecific amplification products or primer dimers. The melting curve helps us determine the specificity of the reaction. Through amplification and melting curve analysis, only complete and specific reactions can provide reliable Ct values 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 Ct is reached 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 , gives the minimum information standards required for publishing an article, and the article is 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 explained in detail.

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

The introduction of fluorescent groups into the system requires an excitation light source to emit light of a certain wavelength. Upon encountering the fluorescent groups, the light reflects light of a different wavelength, which is then received in real time by the receiving device. Because qPCR instruments incorporate these two modules compared to conventional PCR instruments, qPCR consumables have higher requirements than conventional PCR instruments, and their top covers must have excellent light transmittance. During qPCR, do not touch the top cover with your bare hands or while wearing latex gloves. Always 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 method.

The SYBR dye method utilizes the green excitation wavelength of SYBR Green I, a dye that binds to the minor groove of the double helix of all double-stranded DNA (dsDNA), for quantitative analysis. SYBR Green I fluoresces only when bound to double-stranded DNA; free dye molecules do not fluoresce. During the elongation of newly synthesized strands, SYBR Green I is incorporated into the double helix. During denaturation, the DNA double helix unwinds, releasing SYBR Green I and rendering it non-fluorescent. Because nonspecific amplification products and primer dimers are both dsDNA, the SYBR dye method relies solely on primers to ensure specificity. Its advantages lie in its simplicity and low cost, making it suitable for research clients with smaller sample sizes.

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 end is coupled with a quenching group, 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 luminescent group and the quenching group are separated, and the fluorescence signal can be detected.

At the start of the qPCR reaction, the double-stranded template is denatured and melted into single strands via heat. The TaqMan probe preferentially anneals to the template strand, followed by the primer annealing to the template. This single-strand extension proceeds. During this extension process, the Taq enzyme exerts its 5'-3' exonuclease activity, cleaving the probe base by base from the 5' end. The luminescent group separates from the quencher group, allowing the fluorescence detection system to receive a fluorescent signal. Each time a DNA strand is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescent signal is synchronized with the formation of PCR products. The specificity of the TaqMan probe method is not only provided by the primers, but also by the probe molecules. Because of their higher annealing temperature, the TaqMan probe method has greater specificity. By adding multiple probes to a single reaction system, multiple genes can be detected simultaneously.

Molecular beacons are similar to TaqMan probes. In their free state, the probes complement each other to form a hairpin structure, with a luminescent group coupled to the 5' end and a quencher group coupled to the 3' end. In this state, the luminescent group and quencher are in close proximity, and the fluorescence emitted by the luminescent group undergoes fluorescence resonance energy transfer (FRET). However, the signal decays after the quencher is excited. When the reaction system temperature rises, the hairpin structure of the probe opens, and the stem-loop region of the molecular beacon anneals and binds to the template strand. The luminescent group and quencher separate due to their distance, eliminating FRET. The released fluorescence signal is detected by an internal receiver. The newly synthesized complementary strand replaces the molecular beacon, and the beacon molecule, freed from the template strand, reforms the hairpin structure and ceases to emit 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 no non-specific amplification; TaqMan The probe method is suitable for experiments with high specificity requirements, multiple PCR (labeled with different fluorescent groups), SNP Experiments requiring high detection and sensitivity.