Theoretical basis
Polymerase chain reaction has played an important role in the history of biological research as a revolutionary method. Based on this, a number of application technologies including real-time PCR have been developed. Since the birth of real-time PCR technology continues to evolve, from simple amplification to the entire PCR process, real-time PCR shows more sensitive and clear quantitative analysis characteristics and ability to recognize alleles than PCR.
Many people think that real-time means that you can see the growth of each cyclic augmentation curve on the display. This is not the case, and early software was not able to provide a visual augmentation curve during operation. Mainly because SDS software uses the final data of the entire platform to perform data analysis work, rather than analyzing each individual reaction cycle. For some devices, real-time final data must be provided to the analysis software, and some devices do not. The former device allows the software to track the augmentation curve of each sample port in real time and display it on the computer screen. Real-time PCR is actually a real-time device.
RNA quantitative analysis relies on reverse transcriptase to make cDNA (complementary DNA). There are two common reverse transcriptases, AMV and MMLV. AMV is a dimeric protein of the avian myeloblastosis virus, and the MMLV is derived from the monomeric protein of the murine leukemia virus. Both enzymes have RNase to denature RNA into RNA-DNA hybrids, and AMV has higher RNase H activity. RNase H activity and RNA-dependent DNA polymerase activity can be distinguished by mutagenesis. More importantly, each AMV can bring together more molecules and promote the expansion reaction. The native AMV has a higher temperature than the MMLV, 42 ̊C versus 37 ̊C. The modified variants can have higher temperature limits, AMV58 ̊C, MMLV55 ̊C.
According to the above description, it may be considered that the modified AMV is an enzyme suitable for making cDNA from RNA. However, the modified MMLV works better in actual use. The reason for this is still unknown. It is suspected that high temperature destroys the polymerase activity of the two enzymes, but the residual DNA binding activity forms a physical barrier to Taq polymerase. Dimer conformation and higher temperature limits may make AMV perform poorly. For this reason, reverse transcriptase should be used as little as possible in the experiment. It should be emphasized that even without primer cDNA, it can be produced by reverse transcription reaction, and the secondary structure of the RNA template may self-initiate the reaction. There are three ways to prepare a reverse transcription reaction: oligo-dT, random primers or assay-specific primers. Among them, oligo-dT is widely used, and it uses mRNAs as a template. But not all mRNAs have a poly-A tail. The biggest problem is that oligo-dT limits the augmentation region to the vicinity of the mRNA poly-A tail. The reaction begins when the longer fragment is transcribed (<500 bp) at the 3' UTR. Such a result is highly faithful, meaning that the amplified sequence may not cross an exon connection. For some experiments, the 3' UTR is rich in A/T bases that are not conducive to PCR experiments. Therefore, the use of free primers to make transcription sequences abandons the 3' UTR, but free primers also produce cDNAs for ribosomes and transfer RNAs, resulting in large and complex cDNAs. The third method is to use the assay-specific primers, which theoretically meet the requirements of the transcription target sequence to obtain the corresponding cDNA product.
The maximum length of the Real-time PCR amplification is about 250 bases. From the previous discussion, assay-specific primers are the preferred choice for most experiments. However, assay-specific primers have two distinct disadvantages. Each PCR reaction uses more RNA samples and cannot be used to simultaneously process several samples on one instrument. Note that according to the experiment, you need to select the primers reasonably and arrange the experiment plan.
The core of modern PCR reactions is the thermostable DNA polymerase, the most commonly used is Thermus aquaticus, also known as Taq. Wild-type Taq is a polymerase that is based on DNA from 5->3 and has a 3->5 proofreading function. It also has 5'-nuclease activity. Real-time PCR is commonly used to remove the proofreading feature. There are two versions of Taq on the market, a modified Taq and a hot start Taq.
Fluorescent agents are used as signal sources in real-time PCR devices on the market today, and the intensity of fluorescence increases proportionally with the increase of augmented products. Fluorescent molecules absorb photons of narrow wavelengths in light, and the light that can be absorbed by the dye is called the excitation wavelength. The excited molecules are in a higher energy state, the duration is very short, and the molecules rapidly decline to the original state. In this process a photon is emitted at a longer wavelength and has an optimized excitation and divergence wavelength for each fluorescent dye. Fluorescent molecules can be excited or detected in the narrow optimized wavelength range. Fluorescence experiments primarily require that the signal intensity be as large as possible between the first and last 10 PCR cycles. The first fluorochrome dye molecule (donor or reporter) is activated by an externally optimized wavelength ray, releasing a longer wave of light to activate another adjacent molecule. This process is called delta. Molecules that accept light may or may not emit light. Each forwarding process increases the wavelength until the real-time device can receive it. The fluorescers of Real-time PCR are classified into three types according to their functions. 1. The fluorescent signal emitted by Donor is detected during the experiment. 2. The Acceptor is responsible for cooling the signal sent by Donor. 3. It is a reference dye. The reference dye does not react with other components. The software uses it to correct the signals of different wells. In theory, the fluorescer dye can become a reporter. Common dyes are 6-FAM (6-carboxy fluorescein) and YBR® Green I, the former being excited by a wavelength of 488 nm made by an argon ion laser. 6-FAM readily binds to oligonucleotides to release a strong signal. Unlike 6-FAM, YBR® Green I is a free dye.
The cooling molecule can be a fluorescent dye or other molecule capable of absorbing the light energy of the appropriate wavelength. Originally used with 6-FAM is TAMRA (6-carboxy-tetramethylrhodamine). The FAM effectively absorbs photon energy at a location close to the TAMAR. In addition, non-luminescent black dyes can also be used in the reporter. Commonly available are DABSYL (4-(dimethylamino)azobenzene-4'-sulfonyl chloride). The fluorescence signal of the reference group compares the difference of each well, ensuring that the signal of each well is stable overall. The difference in the equipment will result in a deviation of the values between the wells, called the "edge effect", which requires standardization of the values for each well. However, when the edge effect is too large, the gap between the inner sample hole and the outer sample hole is too large to be suitable for the reference group as a basis for standardization. A common reference dye is ROX (6-carboxy-X-rhodamine)
Real-time PCR equipment
It is not wise to introduce the use of all real-time PCRs on the market, but it is necessary to know basic common sense, understand how devices work, device structure and physical limitations. Real-time PCR has 3 major parts, light source: this determines the range of fluorescent dyes, detection system: determines the spectral range and sensitivity. Thermal cycling mechanism: determines the speed of each experiment, the uniform temperature change between samples, and the optimal number of samples.
There are four sources of Real-time PCR, argon ion laser, LED laser, quartz halogen tungsten lamp and xenon lamp. Each different light source determines the working capacity of the device. The main working spectrum of the argon ion laser is 488 nm, which is an efficient wavelength for the reporter. However, at 488 nm under red light, a weaker dye excitation is given, which exceeds the weakening signal at the maximum of 500 nm. A weaker signal limits the utility of the reporter dye. The LED laser emits light at 30-40 nm, and the output energy is weaker than the argon ion laser, but the energy usage is lower. A common source of light is a quartz halogen tungsten lamp that emits a stable wavelength of 360 nm to 1000 nm, covering all visible light, sometimes called "white light." Unlike the lasers mentioned above, two sets of excitation and scattering filters are required to select the wavelength to be used, and some devices use a photomultiplier tube and a CCD camera to capture the signal. The brightness of the xenon lamp is much higher than that of a quartz halogen tungsten lamp, and the wavelength is similar. Five sets of excitation filters and two sets of emission filters were used.
development trend
Real-time PCR presents three development directions
1. The original real-time PCR can only process a single sample, and now more and more devices have a wider range of capabilities, and many devices can provide 5 to 6 unique excitation/diverging filters. In theory, each reaction can allow 5 to 6 different samples to be tested simultaneously. The light emitted by each reporter is separated by physical means.
2. The ability of Real-time PCR equipment to perform thermal cycling has increased, the number of samples that can be loaded has increased, and the improvement in chemical reagents has shortened the time per run, which is the most important performance indicator for high-throughput users.
3. Increase sample yield. The current highest-volume real-time PCR is the ability of the ABI 7900 to load 384 samples at a time, a capacity that exceeds the needs of the general laboratory. But for some users, 1536 sample processing capabilities of higher-production devices become standard.
September 02, 2021
