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Since PCR as a patented biotechnology came onto the stage of molecular biology in 1985, no biotechnology has had such a profound impact on the development of the entire life sciences. There are so many citations and wide 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.
The PCR ( P olymerase C Hain R & lt eaction), translated into Chinese name is "polymerase chain reaction", simply, under the action of two short polynucleotide (primer) and a heat-resistant DNA polymerase, first The DNA template to be amplified is heated, denatured and melted, and then cooled to a certain temperature, the primer is singly combined with the DNA to be amplified, and then the temperature is increased to extend the annealing primer under the action of the DNA polymerase. This denaturation-annealing-extension process is a PCR cycle, which is repeated continuously, and the DNA template can be amplified by 2 n times in just tens of minutes. The entire reaction process is roughly as follows:
The basic reagent composition required in a conventional PCR reaction includes:
Ø DNA template (template), containing DNA fragments to be amplified.
Ø A pair of primers determines the starting and ending positions of amplification.
Ø DNA polymerase (polymerase), copy the region that needs to be amplified.
Ø Deoxynucleoside triphosphate (dNTP), used to construct new complementary chains.
Ø A buffer system containing magnesium ions provides a chemical environment suitable for polymerases to perform their functions.
However, in mature PCR reagents, the reaction reagents other than primers and templates are often configured into Mix according to the optimized reaction parameters. 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 influential molecular biology technology come into being? Here let us expand this history a little bit. Let us return the timeline to 1953 years , that year What happened? New China's first five-year plan? Audrey Hepburn won the Oscar for Best Actress for "Roman Holiday"? Uh, uh , it’s a long way... (ノ｀Д´)ノ Of course, Watson and Crick proposed that DNA is a long chain of double-helical deoxynucleotides paralleled in the opposite direction through complementary pairing of bases! This is this historical photo that has been widely circulated in the biological world:
The one on the left side of the picture above is sitting looking up at Watson. On the right, the one standing on the right and pointing to the model is Crick. ^_^ By the way, Crick died of illness in 2004 , and Watson is still alive. But Crick was twelve years older than Watson...
Based on this structure, they further speculated that this may imply the mechanism of genetic material replication, but this requires experiments to prove. In fact, almost before and after 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 self-replicating molecule and identified the first in 1957. A DNA polymerase, although this enzyme has limited functions, it opens the door to the study of DNA replication mechanisms. 1959 years because Kornberg found that bacterial DNA replication machinery to reproduce in test tubes and DNA replication process and awarded the Nobel Prize in Physiology or Medicine, 1962 year Watson and Crick because DNA double helix model also won the Nobel Prize in Physiology or Medicine.
1969 years, the microbiologist at Indiana University in the United States Thomas Brock and his graduate students Hudson Freeze discovered a thermophilic from volcanic hot springs of Yellowstone National Park in Thermotoga maritima bacteria T.aquaticus (Taq) , for the later 1976 Nian Trela and his Chinese graduate student Qian Jiayun Alice Chien have established a solid foundation (Deoxyribonucleic Acid Polymerase from the Extreme Thermophile Thermus aquaticus) for separation and purification of Taq DNA polymerase to withstand heat >75°C . Gobind Khorana , an Indian scholar who won the Nobel Prize in 1968 for his discovery of genetic code and its function in protein synthesis, and his postdoctoral fellow Kleppe et al. in the 1971 Journal of Molecular Biology (Journal of Molecular Biology) Biology) first published what became known as the "Guide"PCR is technically feasible" article, he wrote in this article:
"A double-stranded molecule wants to obtain two identical structures, and each contains the entire base length of the template strand including primers. DNA polymerase needs to be added to complete such a repair and replication process. Finally, an original double- stranded molecule The molecule produces 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 shifting boy Kleppe clearly proposed the concept of repair replication (repair replication), which was later called the prototype of PCR technology in the academic world, but at that time, sequencing technology was not invented, and thermal stability DNA polymerase was not discovered, and the synthesis of primers was still a scientific performance art, so this idea was quickly forgotten by the academic community. However, a good thought is like a spark, and the scattered sparks always ignite a fire accidentally. In fact, the work of this article as early as 1969 at the Gordon Conference in New Hampshire, Kleppe described to the attendees that a double-stranded DNA molecule produces two double-strands of the same structure. Molecular technology, and the Gordon Conference is like an annual "Huashan Sword" in American academia. It is worth mentioning that there was a professor named Stuart Linn in the audience at the time. In his subsequent teaching, he used these reaction components to demonstrate the experiment according to Kleppe's description. One of the students who attended the class was Karry Mullis, It was he who took the baton of PCR research and completed the final sprint.
In 1979, Sanger published an article titled "Strand Termination DNA Sequencing Method" in the Proceedings of the National Academy of Sciences PNAS. In this article, Sanger mentioned oligonucleotide primers, DNA polymerases, and primers capable of terminating the reaction. The extended and modified nucleic acid can be used in DNA sequencing experiments. Only a year later, in 1980, Sanger again won the Nobel Prize for inventing the Sanger nucleic acid sequencing method. Afterwards, the improved automated Sanger sequencing method achieved the "Human Genomics Project" jointly launched by multiple countries. By 1980, almost the entire academic community knew 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 Southern Blotting probe labeling, this DNA hybridization detection can be used for semi-quantitative detection of target DNA fragments. Everything is conceived and hints at the imminent arrival of PCR technology, just waiting for the appearance of one person, he is Dr. Karry Mullis.
Due to space limitations, let’s only show you the historical moments when PCR inspiration flashes. After all, no matter how much history there is, there will be times when people feel bored... ٩(๑´0`๑)۶ Of course the historical moments Plotting is also essential. In 1972, 28-year-old Karry Mullis received a Ph.D. from the Department of Biochemistry at the University of California, Berkeley, but he did not actually publish a biochemistry-related paper in the six years of doctoral research. Instead, in 1968, he published such a thesis "The Cosmological Significance of Time Reversal" ("The Cosmological Significance of Time Reversal") in Nature magazine 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 shadow of the ivory tower to work in private companies, because this was usually considered the end of a personal academic career (just the same as it is hard to find jobs for biological dogs today). However, Cetus is a very special existence. This company has assembled a group of capable and dreamy scientists. Under the atmosphere of freedom and openness, they are moving towards the set goals. This is similar to the professors and experiments in general colleges at the time. There is a big difference in the way the head of the office does what he does with the door closed. Cetus hired Mullis to take advantage of his expertise in organic chemistry synthesis to synthesize oligonucleotide DNA molecules less than 20 nt in length for research and development in other departments within the company. Mullis later recalled that because the company had already automated operations, his department produced a large number of nucleotide fragments so that the refrigerator had no space to store them, and the department staff were laid back and doing nothing.
Because he was not busy at work, Mullis began to have time to ponder the characteristics of the denaturation and renaturation of the nucleotide fragments he produced. Through continuous experiments, he also figured out a quantitative calculation formula. Based on this, he concluded that if it can increase exponentially, it will become A powerful tool for quickly obtaining large amounts of DNA fragments. It happened that Mullis had been obsessed with how computers deal with logarithmic functions at that time, which led him to link the two things together. Every molecular biologist knows the DNA replication reaction (doubling) and knows the exponential function. 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 the small things continued to double, they would increase very quickly. For example, doubling a specific DNA fragment 30 times will produce 230 copies, so many copies of DNA are enough for researchers to conduct related scientific research. This idea finally broke out one day. According to his own account, it was on a Friday night in May 1983. Mullis drove a silver Honda Civic with his company colleague and his Nth girlfriend, Jennifer (yes, one of his hobbies) It’s a pick-up girl who has four marriages so far.) Travelling from the California Bay Area to a country house in Mendocino County for a pleasant and romantic weekend. Driving on the winding 128 highway in Northern California, he was inspired by the winding roads all the way, and this picture appeared in his mind: The winding mountain highway is a section of DNA double strands, driving The car on the two-lane road is the primer for amplification, and the exhaust from the tailpipe of the car is like a synthesized complementary base sequence. So he stopped the car, woke up his sleeping girlfriend, and explained his thoughts excitedly.
Mullis originally thought that such a simple idea should have been put forward, but after searching the literature, he found no. In the three to five months after the "sudden epiphany," Mullis did not take any action. The reason for this is unknown. However, according to the insider's recollection, either it means that Cetus's raging pace of work makes Mullis have no time to care, or It shows that Mullis has fallen into an endless love, and there is another possibility that it comes from the suspicion and unsupportiveness of colleagues. In August of the same year, Mullis gave a formal report on the principles of PCR in the company for the first time, and the response of the listeners was cold. For one thing, everyone is accustomed to his nonsense; besides, most people think that this principle is too simple. If it is feasible, someone must have done it, otherwise, there must be something that is not feasible, but there is no one. It is clear why it is not feasible.
So Mullis had to set out to prove the feasibility of this idea. Since September 1983, Mullis has carried out some experiments, changing several DNA templates, and also tried different heating and cooling cycles, but the results were not satisfactory. At most, only a looming band was found on the electrophoresis gel. , I cannot convince others that PCR has the effect of amplification.
The effort and time required for any research method to develop from the conception to the practical application stage is mostly underestimated by ordinary people. Since Mullis had not received training in molecular biology before, the company sent technicians to assist, and there were three in total. These people have played an important role in the development of PCR. In November 1984, Mullis technicians obtained credible results for the first time, proving the feasibility of PCR. So at the beginning of 1985, the company decided to send a skilled Japanese technician Randall Saiki. This was a correct decision. Before the advent of automated instruments, PCR was a very cumbersome technology. At the same time, it required many people to be busy in a pile of test tubes, stopwatches, and water baths of different temperatures, and even a separate working space to prevent possible contamination. This requires a long period of repeated operations, and people with unfavorable hands and feet cannot do it. Saiki's results are clean and beautiful, which is convincing.
During the PCR operation, it is necessary to repeat the cycle steps of heating and cooling. The E. coli DNA polymerase used in the previous cycle is denatured and inactivated at high temperature. Therefore, after each cycle reaction, a new one must be added. Polymerase. This approach is not only cumbersome, but also expensive. At the price at the time, the polymerase required for one cycle is worth $1, and 30 cycles are worth $30. Needless to say, more cycles are needed. Therefore, in the spring of 1986, Mullis first proposed the idea of using thermostable enzymes. After searching the literature, I found two related documents. The earlier one was done in the United States, and the other was the work of Russian scientists.
The first report on the isolation of high-temperature DNA polymerase is by Qian Jiayun, a young scientist from Taiwan. In 1973, Qian Jiayun went to the Department of Biology at the University of Cincinnati in Ohio to study. Her tutor J. Trela was curious about a thermophilic bacterium ( Thermus aquaticus , Taq) found in the hot springs of Yellowstone Park, and asked Qian Jiayun and another American student to use the bacterium as the topic of their thesis research. Under the guidance of another teacher, Qian Jiayun successfully isolated the high-temperature resistant Taq DNA polymerase from the bacterium and published the research results in the Journal of Bacteriology in 1976 .
Although Mullis proposed to apply Taq DNA polymerase to PCR, there was no ready-made enzyme available at the time, so he had to find a way to isolate it himself. Cetus has a complete set of equipment for separating proteins, and some people are willing to guide, but Mullis is 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, and purifying Taq DNA polymerase in three weeks. In June 1986, Saiki applied it to PCR for the first time, and the effect was surprisingly good, which can be said to be a hit. Taq DNA polymerase not only greatly simplifies the PCR work, but also has stronger specificity and activity than the previously used E. coli DNase, and the background bands are almost eliminated. Since then, PCR has been a great success. In December 1991, Hoffmann Roche Pharmaceuticals reportedly bought Cetus's PCR technology patent for US$300 million, and Cetus also walked into history. Until recent years, due to previous work published by Qian Jiayun and others, the patent right of Taq DNA polymerase was challenged, and the PCR patent was also affected, but that is another story.
PCR only uses the three steps of "denaturation", "annealing" and "extension" to efficiently amplify a small amount of DNA molecules in vitro, which greatly facilitates the development of molecular biology, so that Bio-Raid A song "The Song of PCR" was specially written for this purpose to celebrate this milestone technological innovation:
Well, the picture feels quite strong, with the lyrics below, it perfectly presents the past and present of PCR^_^
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 came to an end, and then it was serious ╭ ( ￣▽ ￣) ╯ … The driving force behind the continuous update of PCR technology lies in the continuous development of DNA polymerases that synthesize new strands based on templates. In today's diverse PCR applications, it has long been impossible to rely on a single wild-type Taq enzyme to travel the world. Kapa Biosystems company began to adopt the high-throughput "directed evolution" technology concept to design specialized DNA polymerases for special PCR applications, such as NGS. "We screened a large number of special proteins to find mutants that can greatly enhance the function of the enzyme," said John Foskett, technical director of Kapa Biosystems, who specially designed high-fidelity KAPA HiFi DNA polymerase for NGS library amplification. "This product has been optimized several times, which can effectively reduce the preference of PCR amplification and increase the yield, so as to obtain a more consistent sequencing coverage and increase the diversity of the library."
With the continuous development of biological experiment requirements, PCR technology has gradually evolved a series of PCR classifications focusing on different experimental purposes and applications in the course of its development. The more common ones include: touchdown PCR, multiplex PCR, qPCR and ddPCR . Here, we focus on the basic principles of qPCR and its wide range of applications.
Quantitative PCR, also known as real-time PCR, is a DNA amplification reaction that uses fluorescent dyes or fluorescent-labeled probes to detect the total amount of products after each polymerase chain reaction (PCR) cycle, and use fluorescence The form of the signal is recognized by the optical detection system in the qPCR instrument, and finally the method of quantitative analysis of the unknown sample template by drawing the relevant standard curve. Compared with traditional PCR, qPCR can be performed on samples.
After conventional PCR, the PCR products obtained are subjected to agarose gel electrophoresis, which is a simple qualitative analysis.
While qPCR introduces a fluorescent group (dye or probe) into the reaction system, it can be used to label and track the PCR product, real-time online monitoring of the reaction process, combined with the corresponding software to analyze the product, and calculate the template of the sample to be tested. The initial concentration is a fairly accurate quantitative method.
Due to the need for precise quantification of the starting template, there are two specific parameters in the qPCR results that are critical to the evaluation process. The first parameter is the amplification curve. Take the following figure as an example, the abscissa represents the number of cycles, and the ordinate represents the fluorescence intensity or relative fluorescence intensity. At the beginning of the reaction, the fluorescence signal is unstable and fluctuates, and then the signal tends to be stable and exhibits an exponential growth. 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: baseline period, exponential amplification period, and plateau period. After the reaction is completed, the qPCR instrument will generate a threshold line 10 times the standard deviation of the fluorescence signal during the baseline period. The threshold line and the amplification curve will generate an intersection point. The abscissa corresponding to the intersection point represents the Ct value, and the meaning of the Ct value represents each reaction system. The number of amplification cycles experienced when the intensity of the fluorescent signal reaches the threshold 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 used for this analysis because the probe cannot be reduced after being hydrolyzed. It can be seen from the derivation result of the figure below that a peak corresponds to a drop in the fluorescence signal. Each drop represents a large amount of double-stranded product melted within this temperature range. Therefore, a single peak represents only one specific product. A peak represents the presence of non-specific amplification products or primer dimers, and the melting curve helps us to judge the specificity of the reaction. Through amplification curve and melting curve analysis, only complete and specific reactions can the Ct value be true and credible, and can be used for subsequent quantitative calculations.
For subsequent data analysis of qPCR experimental results, the following linear function is usually used for calculation:
Define the initial template quantity as X0 and the product quantity after the nth cycle as Xn, then under ideal PCR conditions, Xn=X0×2n, and under non-ideal PCR conditions, we define the primer amplification efficiency as Ex, Xn=X0×( 1+Ex)n, take the logarithm of both sides at the same time, substitute the Ct value and the product amount X(Ct) when reaching Ct into the finishing formula, lg X0= (- lg(1+Ex) )×C(t)+ lg The final equation of Xc(t) shows that the logarithm of the initial template concentration has a linear relationship with the Ct value. According to the linear relationship, the Ct value can be used to calculate the subsequent expression amount.
In actual scientific research, it is often necessary to consider more complicated parameter indexes than the amplification curve and melting curve. Here is a review published in clinical chemistry, The MIQE Guidelines, which gives the minimum information standards necessary to publish an article. At the same time, the article provides a comprehensive review of qPCR terminology, concepts, research and clinical applications, sample collection, The operating standards and specifications of processing and preparation, nucleic acid quality control, reverse transcription, qPCR process and data analysis have all been elaborated.
Although there are many brands and models of qPCR instruments, they all include the following three reaction modules in terms of working principle: excitation light emission source, receiving device, and PCR reaction module.
Due to the introduction of fluorescent groups in the system, the excitation light emission source is required to emit light of a certain wavelength. When the fluorescent group is encountered, light of another wavelength will be reflected. At this time, it is received by the receiving device in real time. It is precisely because the qPCR instrument is better than the ordinary PCR instrument With the addition of these two modules, qPCR consumables are more demanding than ordinary PCR consumables, and the light transmittance of the top cover must be good. Do not touch the top cover with bare hands or wearing latex gloves when doing qPCR. Be sure to wear PE gloves to prevent impurities from remaining on the top cover to affect 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 utilizes the characteristics of SYBR Green I molecules that can bind to all double-stranded DNA (dsDNA) double helix minor groove regions with green excitation wavelength dyes to achieve quantification. SYBR Green I fluoresces only after it is combined with double-stranded DNA. Free dye molecules do not emit light. SYBR Green I is incorporated into the double-stranded strand during the extension of the newly synthesized strand. The double-stranded DNA is unwound during denaturation and SYBR Green I is released. , No fluorescence. Since the non-specific amplification products and primer dimers are both dsDNA, the SYBR dye method can only use primers to ensure specificity. It has the advantages of simplicity and low cost, and is suitable for scientific research customers with a small sample volume.
The core of the TaqMan probe method is the probe molecule. The TaqMan probe is a single-stranded DNA. The 5'end is coupled with a luminescent group, and the 3'end is coupled with a quenching group. The free intact probe cannot detect the fluorescent signal. , The fluorescence emitted by the luminescent group will be absorbed and quenched by the quencher group, the probe is hydrolyzed, and the luminescent group and the quencher group are far away to detect the fluorescence signal.
At the beginning of the qPCR reaction, the double-stranded template is heated and denatured and melted into a single-stranded strand. The TaqMan probe preferentially anneals to the template strand. The primers are then annealed to the template, followed by single-strand extension. Dicer activity, when encountering the probe, the probe will be removed one by one from the 5'end, and the luminescent group will be separated from the quenching group. Therefore, the fluorescence detection system can receive the fluorescent signal, and each DNA strand is amplified to form one Fluorescent molecules, the accumulation of fluorescent signals and the formation of PCR products 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. Adding multiple probes to a reaction system can do more Simultaneous detection of all genes.
Molecular beacons are similar to TaqMan probes. In the free state, the probes complement each other to form a hairpin structure, with a 5'-end coupling luminescent group and a 3'-end coupling quenching group. In this state, the luminescent group and the quenching group are closer, and the fluorescence emitted by the luminescent group will produce fluorescence resonance energy transfer (FRET), and the signal will attenuate after the quenching group is excited. When the temperature of the reaction system rises, the probe of the hairpin structure is opened, and the stem-loop region of the molecular beacon is annealed and combined with the template strand. After the luminescent group and the quenching group are separated, the FRET phenomenon will not occur due to the long distance. The released fluorescent signal is detected by the receiver device in the machine, and the newly synthesized complementary chain replaces the molecular beacon, and the beacon molecule that breaks away from the template chain re-forms the hairpin structure and no longer releases the fluorescent signal.
Summarize the choice of quantitative methods. In general, in scientific research, most quantitative methods will choose the cheap and convenient SYBR dye method. If there are more stringent quantitative requirements, you can choose the TaqMan probe method; in medical testing, priority Choose an accurate and specific TaqMan probe method. In addition, the SYBR dye method is suitable for reactions with low specificity requirements, reactions with a number of molecules (copy number) exceeding 1000, preliminary experiments before probe experiments, very mature PCR conditions, no dimers, and no non-specific amplification; The TaqMan probe method is suitable for experiments with high specificity requirements, multiplex PCR (labeled with different fluorophores), SNP detection, and experiments with high sensitivity requirements. Molecular beacons due to background fluorescence