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Quality Control of PCR Detection

The quality control of PCR testing is a systematic project, which generally involves three aspects: first, test instruments; second, test reagents and consumables; third, personnel operations. Problems in any one aspect will affect the quality of PCR detection.

Test Instruments

PCR machines and UV gel imaging systems (UV analyzers or portable UV, etc.) and pipettes may affect the quality of PCR assays. The PCR instrument controls the temperature change of the PCR reaction, and the accuracy of its temperature and control time may affect the quality of PCR detection. The accuracy of pipette extraction affects whether the reaction system can achieve the best reaction environment. UV analysis systems have a significant impact on detection sensitivity. There are 3 types of instruments for UV analysis systems: UV analyzers, UV gel imaging systems, and portable UV lamps. The UV analyzer directly observes the DNA amplification band on the agarose gel by eye. The UV gel imaging system is to observe the DNA amplification band on the agarose gel through the camera system on the computer. The portable UV lamp can directly observe the DNA amplification band on the agarose gel during electrophoresis, which is convenient to use and has a relatively low resolution. According to the resolution of these instruments from high to low order is UV analyzer, UV gel imaging system, portable UV lamp. Weak positive amplification bands can sometimes be seen on UV analyzers, but not on UV gel imaging systems. The portable UV lamp can only be seen when the positive amplification band is relatively bright. It is recommended that the portable UV lamp should not be used to ensure the detection quality. It can be used for temporary electrophoresis result observation.

The quality control of the instrument should be implemented through laboratory quality certification, and the validity of the instrument should be carried out on a regular basis. Laboratories without quality certification can regularly ask instrument manufacturers to conduct verification and maintenance to ensure that all instruments are in normal working condition.

2. Test reagents and consumables

In the quality control of PCR detection, the selection of reagents plays a very important role, and it is also the detection quality control factor that experimenters pay the most attention to. Reagents must be selected from manufacturers with good quality and reputation. Once selected, it is best not to change them easily. If it must be replaced, a strict comparison test should be carried out with the original product, including tests of sensitivity, specificity, and shelf life of reagents. And often pay attention to the differences in the quality of different batches of products. For example, the quality of TaqDNA polymerase produced by different manufacturers is different. Although they are all marked with the same activity unit, the difference in the calibration method, the composition of the preservation solution, and the transportation process often lead to differences in the actual activity unit. Sometimes, the actual activity unit is different. Due to its low activity, weak positive samples are missed; sometimes non-specific amplification occurs due to its high activity. This is similar to the role of enzyme conjugates in ELISA assays. In fact, there are many reagents that affect the results of PCR detection. It can be said that all the reagents used in PCR detection may affect the detection effect. Controlling the quality of reagents is a prerequisite for PCR detection. We believe that it is best to choose PCR kits to control the quality of the reagents.

To evaluate a PCR kit, in addition to considering its sensitivity, specificity, stability, and ease of operation, it should also include: the extent to which the reagents provided by the kit cover the entire PCR detection test. If the kit provides the reagents for the entire PCR detection test, it indicates that the kit has the whole-process control performance for the reagents of the PCR detection test. On the contrary, the quality control degree of the reagents for PCR detection is relatively low, which means that the quality control degree of the detection is low. For example, ethidium bromide only acts as a fluorescent dye. If it fails under sunlight for a long time, the fluorescence efficiency will decrease under ultraviolet light, which may cause some weak positive samples to be missed and false negatives. Problems with other reagents also affect the quality of the test. During the production process of the kit, there is a set of reagent production quality control system. Each reagent component has been tested for sensitivity, sensitivity and specificity quality control, and the quality of each reagent can be guaranteed within the validity period. Therefore, choosing a kit for PCR detection is an ideal control method. In PCR detection, it is best to use all kits to provide reagents, which is beneficial to the quality control of PCR detection.

Experimental consumables are also one of the factors that affect the quality of PCR detection. The thickness of the PCR reaction tube and the heat conduction performance directly affect the PCR detection effect. Mineral oil generally does not need to be added to the hot-lid PCR instrument, but some PCR reaction tubes are not tightly capped, resulting in the evaporation of water in the reaction solution during the amplification process, which seriously affects the detection sensitivity. The PCR reaction system is a small amount. When the production quality of the tip of the pipette is not good, sometimes the liquid residue is large or the flooding effect is strong, resulting in insufficient reagent volume in the kit and inaccurate configuration of the PCR reaction system, resulting in a decrease in the detection quality. .

In each PCR test, a negative control sample and a positive control sample must be set up. When the negative control sample is detected as negative, it indicates that the reagents in the whole process of the test are not contaminated; when the positive control sample is detected as positive, it indicates that the working system of DNA extraction (RNA extraction, RNA reverse transcription), DNA amplification and electrophoresis identification is normal. The test samples can only be judged on the premise that the test results of the negative control samples and positive control samples are established. Therefore, each test should set up controls indicating DNA extraction (RNA extraction, RNA reverse transcription), DNA amplification and electrophoresis identification. Only by setting up controls throughout the test can the test results be proven. This is very important for PCR and RT-PCR detection assays.

A positive control is a must-have component in the kit. There are three types of positive controls: the first is to use the detected pathogenic microorganisms as positive controls. The advantage of such a direct comparison is that it is intuitive, accurate, and the condition of the experiment is complete, which can indicate whether the experiment is established. The disadvantage is that it increases the index of PCR contamination during the detection process. In addition, it cannot be shown that each test sample control holds. The second is to set up a microorganism not related to the detection of the virus as a positive control. The advantage is that the risk of PCR contamination is reduced and the biosafety of the kit is improved. The disadvantage is that it is only a reference to the positive control, which is not directly and completely reflects the establishment of this test, nor can it show that the control of each test sample is established. It is very suitable for the detection of pathogenic microorganisms in laboratories that are afraid of contamination or requiring high biosafety levels. The third is to set up a reference control in each reaction tube. In addition to the positive amplification band, another amplification band is set up to indicate the detection situation in each reaction tube. When the test is positive, there are two amplified bands of different sizes, and when the test is negative, one amplified band appears. This kind of control establishment has high technical requirements and high cost, and the control effect is the most ideal, which can eliminate the influence of the operation error of each test sample or the problem of the reagent on the test. Since two templates are amplified at the same time in one reaction tube, there is a certain amount of interference between each other, so the sensitivity of the detection of pathogenic microorganisms is more or less affected. This control method may become the development trend of PCR control.

1. 3. Personnel Operation

Several of our laboratory personnel have tested the same sample at the same time, and the test results do vary. Frequent testers are 10 to 100 times more sensitive than other testers. It shows that the PCR detection test does have certain operating techniques that need to be familiar with and trained, and different personnel have a certain impact on the test results. It is necessary to strengthen the training of personnel's operating skills, and it is necessary for laboratory technicians to have a proficient process for PCR detection.

PCR contamination control is a content that PCR testing operators must pay great attention to. The laboratory is set up in the distribution area, template extraction area, amplification area, and electrophoresis area. The logistics should be in the order of distribution liquid area, template extraction area, amplification area, and electrophoresis area, and backflow is strictly prohibited. Personnel operation should also be very careful, such as regular cleaning and disinfection, and regular cleaning and disinfection of pipettes. Timely test operation is simple, fast and accurate.

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Common PCR and QPCR primers similarities and differences

The same:

• The search of the sequence is consistent;

• The sequence selection should be in the conserved segment of the gene;

• Choose the appropriate amplified fragment size

• Avoid the formation of 4 or more consecutive pairs of primers themselves or with primers;

• Avoid the primer itself forming a circular hairpin structure;

• Tm value is 55-65℃, GC content is 40%-60%;

• The TM difference between primers should not exceed 2°C;

• Avoid 3 or more consecutive identical bases at the 3' end of the primer;

Difference:

Real time PCR primers

• Length of PCR product; real-time PCR requires within 300bp, generally 80-150bp is preferred;

• Multiple pairs of target genes are amplified at the same time, and the primer conditions found in the literature will be different. It is necessary to design primers with the same conditions as possible;

• When the target gene content is relatively low, it is necessary to design primers with relatively high sensitivity;

• Relative to electrophoresis, Real time PCR has higher sensitivity, higher requirements for primers, and fewer primer dimers; melting curve requires a single product;

• The purpose of Real time PCR is to perform quantitative or relative quantification, which has requirements for the efficiency of amplification; high requirements for the secondary structure of primers;

Common PCR primers:

• Depending on the requirements of the experiment, the length is generally from 150bp to several thousand bp;

• The requirement for secondary structure is not as high as that of real time PCR;

• The requirement for amplification efficiency is not high;

• Gradient PCR instrument can be used, and different annealing temperatures can be selected, and the requirements for primer annealing temperature do not need to be consistent;

Different when verifying:

Primer specificity (same point):

Ø The specificity of primers is guaranteed by blast before use;

Difference: real time PCR

Ø Verified by the melting curve in the experimental results;

Ø The specific product peak of PCR should be within two degrees of the PCR product in the primer report;

Ordinary PCR identifies the specificity of the product by the length of the product and running the band;

Real time PCR can identify the relative amount of products through amplification curve and melting curve analysis, and can also perform electrophoresis analysis on the final product, which is more comprehensive and scientific!

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The method and characteristics of PCR identification kit

PCR Identification Kit Features:

1. One-tube operation, users only need to provide samples.

2. Primers are designed according to the conserved region of soybean heat shock protein gene, which can specifically detect soybean components in the sample, but cannot detect other non-soy components.

3. Fast, the entire detection process (according to one sample) takes only 2.0 hours.

4. Only need common PCR machine and gel electrophoresis machine, no need to configure expensive equipment.

5. The lower limit of detection for soybean components in mixed samples is 0.1%, and the lower detection limit for nucleic acids for soybean components in samples is 1.0ng/μL.

6. This product can only be used for scientific research, enough for 50 times of routine PCR in 40uL system.

How to use PCR identification kit:

1. Dilute the standard curve samples (take 10E2-10E7 copies/μL as an example of six 10-fold dilutions). Since the standard concentration is very high, the following dilution operations must be performed in a separate area, and must not contaminate the sample or other components of the kit).

1.1 Label the 6 centrifuge tubes as 7, 6, 5, 4, 3, and 2.

2.1 Use a cored pipette tip to add 45 μL of fluorescent PCR template dilution solution, *use a cored pipette tip, the same below).

3.1 Add 5 μL of positive control (concentration of 1×10E8 copies/μL, provided by the kit) to the No. 7 tube, shake well for 1 minute, and obtain a standard curve sample of 1×10E7 copies/μL. Set aside on ice.

4. 1Change the pipette tip, add 5 μL of 1×10E7 copies/μL positive control (obtained in the previous step) to tube 6, shake well for 1 minute, and obtain a standard curve sample of 1×10E6 copies/μL. Set aside on ice.

5.1 Change the pipette tip, add 5 μL of 1×10E6 copy/μL positive control (dilution in the previous step) to tube 5, and shake well for 1 minute

clock to obtain a standard curve sample of 1×10E5 copies/μL. Set aside on ice.

6. 1Repeat the above procedure until 6 dilutions of the standard curve samples are obtained. Set aside on ice.

2. Preparation of sample DNA

2.1 Purify the DNA of the sample by the method of your choice. This product is compatible with most nucleic acid purification products on the market.

2.2 If there are N samples, N+2 sample extractions need to be performed, one extra for the sample preparation positive control tube and the other for the sample preparation negative control tube.

2.3.Set up the qPCR reaction (20μL system, in the sample preparation room)

2.4. If doing quantitative analysis and only doing 1 replicate, label N+9 PCR tubes, of which N+2 are for the N+2 samples from the previous step and 1 is for the PCR negative control (use water as template). ), 6 for the standard curve. If qualitative analysis is performed and only 1 repetition is performed, label N+4 PCR tubes, of which N+2 are used for the N+2 samples obtained in the previous step, and 1 is used for PCR negative control (use water as template) , 1 for PCR positive control.

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Key parameters and important characteristics of fluorescent quantitative PCR instrument

When purchasing a fluorescent quantitative PCR instrument, several key parameters should be paid attention to:

1. The detection throughput of the instrument (Throughput)

The first thing to consider is the frequency with which the laboratory currently needs to use a quantitative PCR instrument. David Ginzinger, head of genome analysis equipment at the University of California, San Francisco Cancer Center, has tested most of the quantitative PCR machines on the market. He said that if it is not used in large-scale batches, for example, it is mainly used to test known genes, then expensive high-end products are really not needed. But if it is those laboratories that need to find new drug targets and disease markers, they need instruments that can accommodate 384-well plates and run faster. However, just such a high-throughput instrument is not fast enough, and you will find that sample preparation becomes a bottleneck for the speed limit. Therefore, high-throughput laboratories often need further automated instruments. As long as the "click", the instrument will automatically process samples, purify nucleic acids, and prepare PCR templates.

2. The ability of multiple amplification

Multiple amplification is getting hotter and hotter. Real-time quantitative PCR machines are not immune. The high-end quantitative PCR instrument has multiple detection channels, so the instrument can detect the amplification process of multiple templates in the same sample tank. The new LightCycler2.0 has 6 detection channels, 3 more than the original. SmartCycler II has 4 detection channels. Stratagene, famous for its library

products, also has a quantitative PCR machine Mx3000P, which is less than half the price of Roche and has a good reputation, which is 4 detection channels. However, multiple amplification is not suitable for everyone, because it complicates the experiment.

3. software

When you are going to choose a quantitative PCR machine, you must check the accompanying software. Is it easy to use? Is it possible to perform the analysis you need? There are several output formats. However, you need to pay attention to whether the software that is too easy to use has simple functions and cannot perform complex calculations. The previous software still requires you to do further analysis. The new software can already handle everything. In addition to basic functions, some software can also have more functions, such as primer and probe design, multiple analysis design, background correction, data normalization, and so on. Willian Demyan, head of the Application and Technical Services Department of Roche's Applied Science Department, emphasized: Quantitative PCR is a geometric amplification process. At the beginning, the difference between one copy and the result is geometrically different.

4. Flexibility

Although the large-scale busy laboratory equipment is advanced, it is enviable, and the conventional laboratory can also have wonderful choices. The current suppliers have many technical experts, not only understand your current needs, but also consider possible changes in your future requirements very thoughtfully, such as providing a variety of upgradeable accessories and modules. BioRad's MyCycler can select modules and upgrade to real-time quantitative. ABI's quantitative PCR instrument has a variety of different sample tanks, which can change a 96-well tank into a double 384-well tank to meet part of the high-throughput requirements. For laboratories that are just starting to do a small amount of qPCR, the SmartCycler II quantitative PCR instrument is a good choice. A basic module has 16 independent temperature-controlled sample tanks, and 16 completely independent different experiments can be performed at the same time or before and after. Access"; when the laboratory needs a larger-scale experiment, it can be upgraded economically, from 1 to 6 basic modules, that is, 96 sample wells. Although a 96-well plate cannot be used, when a student starts a small experiment, others can plug in and start their own independent experiments at any time without interfering with each other, which increases flexibility. It is a very flexible choice for laboratories that only do a few experiments at the beginning and have a tight budget.

5. Uncertainty

 Although the software can be partially corrected, it is better to buy an instrument with less system error and not require so many calculation corrections than to rely on calculation corrections. Cinzinger believes that special attention should be paid to the differences between the sample holes of the instrument. In addition to the well-known temperature difference between the sample holes, there are also the optical path distance between the sample hole and the detection probe, the slight difference in the length of the optical fiber responsible for receiving and transmitting signals, the slight difference in the distance and angle between the lens and the sample hole, and even The influence of the sample slot on signal transmission. Cinzinger has done the same test on most of the commercially available quantitative PCR machines. He performs real-time amplification of the same samples and reaction reagents in each well of the instrument. Unfortunately, many times, there will be different results between different sample wells.

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Four elements of qPCR performance analysis-efficiency, linear dynamic range, detection limit and precision

When conducting qPCR experiments, the following detection performance characteristics must be determined: PCR efficiency, linear dynamic range, LOD and precision.

1. The efficiency of PCR:

Powerful and accurate qPCR assays usually have high efficiency. When reporting the mRNA concentration of the target gene relative to the reference gene, PCR efficiency is very important. The ΔΔCq method is one of the most common methods for determining the concentration difference between a sample and a single reference gene used for normalization. If the difference (ΔCq) between the Cq values of the target gene and the reference gene is calculated, the ΔCq values of different samples can be directly compared. It is worth noting that the comparison of two genes must be carried out under similar amplification efficiency. However, the most common method is not necessarily the most appropriate. On the contrary, more general quantitative models have been developed to correct for differences in amplification efficiency and the use of multiple reference genes.

The PCR amplification efficiency must be established by the calibration curve method, because the calibration method is simple, fast, and reproducible, which ensures the average reaction efficiency, analytical sensitivity and robustness of the detection method of PCR. The amplification efficiency is calculated by the slope of the linear part of the calibration curve. The specific calculation formula is: PCR efficiency = 10-1/slope-1, that is, the logarithm of the initial template concentration is taken as the X axis (independent variable), and the Cq value is Plot the Y axis (dependent variable). Theoretically, the maximum efficiency is 1.00 (or 100%). This value indicates that the product quantity per cycle is doubled. Ideally, the reported CI (confidence interval) or SE (error) value of the average PCR efficiency should be obtained by the two-time calibration curve method.

When publishing an article, in addition to submitting the calibration curve of each quantitative target, the slope of the calibration curve and the intercept on the Y-axis must also be provided. Differences in PCR efficiency will produce calibration curves with different slopes. As the amount of template changes, the difference between the Cq value of the target gene and the reference gene is not fixed. Therefore, the relative concentration calculated based on the fixed Cq value is inaccurate and may produce misleading results.

A Cq value greater than 40 is suspicious, and it is not recommended to report it due to its low efficiency. However, this arbitrarily setting Cq threshold is unscientific, because these values may be low (can eliminate valid results) or high (can increase false positive results).

2. Linear dynamic range:

The dynamic range of the PCR reaction should be linear, that is, the highest or lowest quantitative copy number should be obtained by the average calibration curve method, and the submitted article should include the linear dynamic range. The generation of the calibration curve depends on the template used, and the dynamic range should span at least 3 orders of magnitude, preferably to 5 or 6 log10 concentration ranges. The linear interval of the calibration curve must include the quantitative range of the target nucleic acid. Since the definition of the lower limit of quantification is often confusing, the variation at the lowest concentration in the so-called linear range in the submitted article should be reported. In addition, the linear correlation coefficient (R2 value) must be reported, and it is best to provide the CI value over the entire linear dynamic range.

3. Detection limit:

The detection limit is defined as the lowest concentration that can detect 95% of positive samples. In other words, if a sample to be tested with a concentration at the LOD level is repeated multiple times, the probability of an invalid result is less than 5%. The occurrence of low-copy PCR is random and limited, and it is impossible for the LOD value of a single PCR to be less than 3 copies. However, if multiple reactions are performed, accurate quantification of the low concentration range can be obtained by digital PCR. In fact, the concentration calibrator can be subjected to limiting dilution, and the failure rate and success rate of the PCR reaction can be calculated according to the Poisson distribution.

4. Precision:

There are many factors that cause qPCR variation, including temperature differences that affect annealing and denaturation, concentration variations caused by sampling errors, and random errors. The precision of qPCR is mainly affected by concentration and decreases with the increase of copy number. It is best to perform multiple replicates, and mark the intra-assay variation (repeatability) in the form of SD error bars and the CI (confidence interval) of the calibration curve in the graph. The coefficient of variation CV cannot be used to describe Cq, but it can be used to express the variation of copy number and concentration. The variation caused by technology should be distinguished from the biological variation. Biological replication can directly lead to significant differences in qPCR results between groups or between different treatment methods. For diagnostic analysis, it is also necessary to report the inter-batch precision (reproducibility) of different locations and different operators.

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What value does the real-time quantitative PCR instrument provide in medicine?

Since its birth, the real-time quantitative PCR instrument detection technology has been more and more favored by laboratory teachers. It is a method that uses fluorescent chemicals to measure the total amount of products after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction. A method for quantitative analysis of specific DNA sequences in the sample to be tested by internal reference or external reference method.

Medical research on real-time quantitative pcr instrument:

1. Prenatal diagnosis:

People are still unable to treat genetic diseases caused by genetic material changes. So far, only prenatal monitoring can be used to reduce the birth of sick babies to prevent the occurrence of various genetic diseases. For example, in order to reduce the birth of children with X-linked genetic diseases, separating fetal DNA from the peripheral blood of pregnant women and detecting the Y sex-determining region gene by real-time fluorescent quantitative PCR is a non-invasive method that is easily accepted by pregnant women.

2, pathogen detection:

Fluorescence quantitative PCR detection technology can detect Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, human papilloma virus, herpes simplex virus, human immunodeficiency virus, hepatitis virus, influenza virus, Mycobacterium tuberculosis, Epstein-Barr virus and cytomegalovirus, etc. The pathogen is quantitatively determined. Compared with traditional detection methods, it has the advantages of high sensitivity, less sampling, quickness and simplicity

3. Evaluation of drug efficacy:

Quantitative analysis of hepatitis B virus (HBV) and hepatitis C virus (HCV) shows that the amount of virus is related to the efficacy of certain drugs. HCV is expressed at high levels and is not sensitive to interferon treatment, while HCV is low in titers and interferon is sensitive; during lamivudine treatment, the serum level of HBV-DNA has declined, and then if it rises again or exceeds the previous level , It indicates that the virus has mutated.

4. Tumor gene detection:

Although the mechanism of tumor pathogenesis is not yet clear, it has been widely accepted that mutations in related genes are the root cause of carcinogenic transformation. Increased expression and mutation of oncogenes can appear in the early stages of many tumors. Real-time fluorescent quantitative PCR can not only detect gene mutations effectively, but also accurately detect the expression of oncogenes. At present, this method has been used to detect the expression of multiple genes such as telomerase hTERT gene, chronic myelogenous leukemia WT1 gene, tumor ER gene, prostate cancer PSM gene, tumor-related viral genes and so on.

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The common faults of automatic gradient PCR instrument mainly include the following categories

The primers added in the PCR amplification of the automatic gradient PCR instrument are labeled with isotopes, fluorescein, etc., and the primers and fluorescent probes are used to specifically bind to the template at the same time for amplification. The results of the amplification are connected to the computer analysis and processing system through the real-time acquisition signal of the fluorescence signal acquisition system, and the quantified real-time result output is obtained. Multi-fluorescence markers and target gene expression products can be detected, because only one target gene amplification amount can be detected at a time, and multiple amplifications are required to detect the amount of different target gene fragments. Multi-channel facilitates multiplex PCR and realizes the function of detecting multiple target genes at one time.

The automatic gradient PCR instrument is an instrument that is used in the laboratory with a very high frequency and time, so the failure rate is relatively high. The faults of the thermal cycler mainly include the following categories:

1. Refrigeration semiconductor failure

The refrigeration semiconductor is the core component of the PCR machine. The refrigeration semiconductor used in the PCR machine is a semiconductor chip dedicated to the industrial PCR machine, which is mainly able to withstand large temperature changes. However, the production of these refrigeration semiconductors is semi-manual, and there is no good way to test their durability. In short, there is a certain failure rate.

The number of temperature changes resistance of refrigeration semiconductors is related to their materials and production technology. Such as ceramic chip dicing, non-hard welding and other technologies. Good refrigeration semiconductor manufacturers will provide experimental data on the number of temperature changes. Its performance will decrease with the number of temperature changes. Some PCR machines have the function of counting the number of temperature changes.

2. The temperature sensor is faulty

There are roughly three types of temperature sensors used in PCR machines, namely thermal resistance type, thermocouple type and semiconductor type. Thermal resistance type and thermocouple type are used more and have a lower failure rate, while semiconductor type has a built-in circuit. The failure rate is high when used in the instrument (of course it has other advantages). Some PCR machines will report sensor failures. In fact, the sensor, sensor-related wiring and sensor signal processing circuit failures are all possible.

3, control board failure

The control board is the command center of the PCR instrument, and it is more susceptible to failures caused by external factors, such as excessive dust causing excessive temperature of the control board, excessive humidity causing short circuits, crosstalk (from the power cord), and electromagnetic radiation (Wireless) interference, etc.

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How to improve the sensitivity of RT-PCR reaction system

1. Isolate high-quality RNA

Successful cDNA synthesis comes from high-quality RNA. High-quality RNA should be full-length and free of reverse transcriptase inhibitors such as EDTA or SDS, and minimize genomic DNA contamination. The quality of RNA determines the maximum amount of sequence information you can transcribe to cDNA.

2. Use reverse transcriptase without RNaseH activity

RNase inhibitors are often added in the reverse transcription reaction to increase the length and yield of cDNA synthesis. Both M-MLV and AMV have endogenous RNaseH activity in addition to their own polymerase activity. RNaseH activity and polymerase activity compete with each other for the hybrid strand formed between the RNA template and the DNA primer or cDNA extension strand, and degrade the RNA strand in the RNA:DNA complex. The RNA template degraded by RNaseH activity can no longer be used as an effective substrate for cDNA synthesis, reducing the yield and length of cDNA synthesis. Therefore, eliminating or greatly reducing the RNaseH activity of reverse transcriptase will be of great benefit.

3. Increase the reverse transcription temperature

A higher holding temperature helps to open the RNA secondary structure and increases the yield of the reaction. For most RNA templates, in the absence of buffer or salt, the RNA and primers are incubated at 65°C and then quickly cooled on ice to eliminate most of the secondary structure so that the primers can bind.

4. Additives to promote reverse transcription

Additives including glycerol and DMSO to the *strand synthesis reaction can reduce the stability of the nucleic acid double-strand and untie the RNA secondary structure. You can add 20% glycerol or 10% DMSO without affecting SuperScriptⅡ or M- MLV activity. AMV can also tolerate 20% more glycerol without reducing activity.

5. RNaseH treatment

Treatment of cDNA synthesis reaction with RNaseH before PCR can increase sensitivity. For some templates, the RNA in the cDNA synthesis reaction prevents the binding of the amplified product. In this case, RNaseH treatment can increase sensitivity. Generally, when amplifying longer full-length cDNA target templates, RNaseH treatment is necessary, such as low-copy tuberous scherosis II. For this difficult template, RNaseH processing strengthened the signal generated by SuperScriptⅡ or AMV synthesized cDNA. For most RT-PCR reactions, RNaseH treatment is optional, because the PCR denaturation step incubation at 95°C generally hydrolyzes the RNA in the RNA:DNA complex.

6. Improvements in detection methods for small amounts of RNA

When there is only a small amount of RNA, RT-PCR is particularly challenging. The glycogen as a carrier added during the RNA isolation process helps increase the yield of small samples. You can add RNase-free glycogen at the same time as Trizol. For a sample of 106 cultured cells in tissues less than 50 mg, the recommended concentration of RNase-free glycogen is 250 μg/ml. Adding acetylated BSA to the reverse transcription reaction using SuperScript Ⅱ can increase the sensitivity, and for small amounts of RNA, reducing the amount of SuperScript Ⅱ and adding 40 units of RnaseOut nuclease inhibitor can increase the detection level. If glycogen is used in RNA isolation, it is still recommended to add BSA or RNase inhibitor when using SuperScriptⅡ for reverse transcription reaction.

7. Reduce genomic DNA pollution:

A potential difficulty encountered by RT-PCR is that there is genomic DNA contamination in the RNA. The RNA can be treated with amplification-grade DNase I to remove the contaminated DNA before reverse transcription. The sample was incubated in 2.0 mM EDTA at 65°C for 10 minutes to stop the DNase I digestion. EDTA can chelate magnesium ions and prevent the magnesium ion-dependent DNA hydrolysis that occurs at high temperatures. In order to separate the amplified cDNA from the amplified product of the contaminated genomic DNA, primers that anneal to the separated exons can be designed. PCR products derived from cDNA will be shorter than products derived from contaminated genomic DNA. In addition, a control experiment without reverse transcription is performed on each RNA template to determine whether a given fragment is derived from genomic DNA or cDNA. The PCR product obtained without reverse transcription is derived from the genome.

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If you want to maintain the gradient PCR gene thermal cycler, you must pay attention to these matters!

 Gradient PCR gene amplification instrument is an instrument and equipment that uses PCR (polymerase chain reaction) technology to amplify specific DNA. It is widely used in medical and biological laboratories, for example, to determine whether a sample will show certain Atlas of genetic diseases, diagnosis of infectious diseases, gene duplication, and paternity testing, etc.

PCR (polymerase chain reaction) is a method of enzymatically synthesizing specific DNA fragments in vitro. It consists of several steps of high temperature denaturation, low temperature annealing, and temperature extension. It has the characteristics of strong specificity, high sensitivity, simple operation and time-saving.

The main function of PCR gene amplification instrument is similar to the basic measuring instrument, and it has higher requirements for measurement elements. Once out of control, the instrument will not work normally, so the PCR gene amplification instrument also needs regular maintenance.

Problems that need attention in the maintenance of gradient PCR gene amplification instrument:

1. The PCR gene amplification instrument needs to be tested regularly, usually at least once every six months.

2. The required temperature of the PCR reaction is inconsistent with the actual distribution of the reaction temperature. When the average temperature difference of each well is found to deviate from the set temperature by more than 1 to 2°C, the temperature correction method can be used to correct the actual PCR reaction temperature difference.

3. The key to the PCR reaction process is the time control of the rising and cooling process. The shorter the requirement, the better. When the cooling process of the PCR machine exceeds 60s, the refrigeration system of the instrument should be checked, and the reaction base should be cleaned for the air-cooled PCR machine. For other refrigeration systems, check the relevant refrigeration components.

4. In general, if the temperature correction method can be used to correct the temperature of the gradient PCR gene amplification instrument, do not open or adjust the electronic control parts of the instrument easily. If necessary, please ask the maintenance personnel to repair or use the detailed drawings of the electronic circuit of the instrument for maintenance.

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Optimization of conventional PCR reactions

A. DNA template:

Try to use high-quality, purified DNA as a template

· When the fidelity needs to be improved, a higher DNA template concentration can be used and the number of cycles can be reduced

· Template dosage: Take 50 μl reaction system as an example——

Human genomic DNA: 0.1~1.0 μg

E. coli genomic DNA: 10~100 ng

Lambda DNA: 0.5~5 ng

Plasmid or viral DNA: 0.1~10 ng

B. Primer design principles:

· The length of the primer must meet specificity requirements, generally between 18 and 25 bases; when amplifying long fragments, it is best to be between 24 and 30 bases;

· When introducing a cloning site, the end of the primer should add more than 3 bases;

· The (G+C)% content should be controlled within 40-60% as much as possible, and the (G+C)% content of the two primers should be as close as possible;

· GC bases are evenly distributed in primers;

· Try to avoid the same base appearing more than three times in a row, and avoid using A or T at the 3'end;

· Avoid primer internal pairing to form a secondary structure;

· Avoid pairing bases between the forward and reverse primers, especially the three bases at the 3'end, otherwise it is easy to generate primer dimers;

· The melting temperature (Tm) of the two primers should be between 42~65℃, and the difference between the two primers should not exceed 5℃;

· Calculation method of primer Tm value:

Below 20 nt: Tm = 2℃ x (A + T) + 4℃ x (G + C)

20 nt or more: Tm = 81.5 + 0.41 x (GC%) -600/nt (nt: base number of primer)

· Primer usage:

· 0.1~1.0 μM, usually starting at 0.2 μM, adjust the dosage according to the system;

· When using degenerate primers or random primers, the total amount of primers needs to be increased to compensate for the loss of yield; however, as the amount of primers increases, the specificity will decrease;

· When the template is larger or larger, or the structure is more complicated (such as human genomic DNA), you need to reduce the amount of primers to improve specificity;

· When the template is small (such as a plasmid template), increasing the amount of primers can increase the yield.

C. Nucleotides (dNTPs):

· The concentration of conventional dNTPs is 0.1~1.0 mM for each nucleotide, usually starting at 0.2 mM, and the dosage can be adjusted according to the system;

· Low concentration (0.05~0.1 mM) can increase fidelity, but will reduce yield;

· High concentration increases yield, especially long-segment PCR, but reduces fidelity.

D. Magnesium ion concentration

· For Taq DNA polymerase, the optimal concentration of magnesium ions is 1.5~2.0 mM;

· The optimal concentration depends on the template, buffer, DNA and dNTPs (each of which may chelate magnesium ions);

· Too low magnesium ion concentration will reduce output;

· Too high magnesium ion concentration will increase non-specific PCR products;

· When optimizing the magnesium ion concentration, it is usually increased by 0.5 mM gradient, up to 4 mM.

E. Taq DNA polymerase concentration

· The recommended concentration is 1~2.5 U/50 μl reaction system.

F. Initial reaction

· Prepare the reaction system on ice;

· Add polymerase at the end;

· After preheating the thermal cycler to the denaturation temperature (94°C), put it in the PCR tube and proceed immediately.

G. Denaturation temperature and time

· The initial denaturation is usually at 94°C, so that the DNA double strands are completely opened;

· Denaturation time is usually 15~30 seconds;

· Avoid prolonged or high temperature incubation;

· Templates with high GC content can increase the denaturation temperature to 98°C.

H. Annealing temperature and time

· Normally, the annealing temperature is the primer Tm minus 5°C, which is between 55 and 60°C;

· Increasing the annealing temperature is conducive to reducing non-specific bands;

· The conventional annealing time is 15-30 seconds.

I. Extended temperature and time

· The extension reaction is usually carried out at 72°C.

· The extension time of Taq enzyme is about 15~30 seconds/kb DNA;

· When the product is less than 1 kb, the recommended extension time is 30 to 60 seconds;

· When the product is larger than 3 kb or the reaction exceeds 30 cycles, a longer extension time may be required.

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Analysis of Several Major Factors Affecting the Uniformity of 96-Well PCR Machine

1. The unevenness of refrigeration semiconductor chips

Most of the current PCR machines use semiconductor refrigeration chips to control the temperature. When it is energized, one side is hot and the other side is cold, and the opposite electrode is opposite to the hot and cold side.

Although the semiconductor refrigeration film is industrialized production, almost all of them are produced manually, so each film is different, and large manufacturers will match them according to the needs of users. For example, if a PCR machine requires three pieces, then three pieces will have roughly the same resistance value. The resistance value is generally written by the manufacturer on each refrigeration chip (the measurement is not accurate when using a multimeter by yourself, because the temperature changes when the power is turned on, and the resistance value will change), so the quality of the semiconductor chip will affect the uniformity of the PCR Machine.

2. Number of temperature control points

On the one hand, there is a certain difference in the semiconductor chip itself, on the other hand, the performance will decrease after a period of use, but the degree of decline is not consistent, and the heat dissipation of the opposite side of the edge and the middle semiconductor chip is inconsistent (to be discussed later). Cause the uneven temperature of the module. The commonly used solution is to place a temperature sensor under each semiconductor chip, and have a corresponding control circuit to independently control and calibrate (this will increase the cost of the manufacturer). For example, AB's 9700 has three sensors and corresponding circuits, while the low-end has only one.

Another advantage of multiple sensors is that different temperatures can be controlled separately to generate temperature gradients.

3. Edge effects and mitigation methods

The edge of the module (referring to the metal block where the PCR tube is placed) dissipates heat quickly, and the temperature is generally low, which is the so-called edge effect. In fact, the radiator also has the problem of low edge temperature. To reduce the edge effect, the edge-assisted heating technology is generally used, that is, a circle of resistance wire or heating film for auxiliary heating is added to the periphery of the module. But in fact, the edge effect is not uniform. One is that the temperature at the corners is lower, and the other is also related to the blowing direction of the radiator fan (front and back blowing is uneven front and back, while blowing from bottom to top is uneven center and edge).

4, the heat dissipation uniformity of the radiator

The heat dissipation uniformity of the radiator has a great influence on the temperature uniformity of the module. The semiconductor chip controls the temperature difference between its front and back sides, and each semiconductor chip is controlled as a whole. If the temperature of the heat sink on the back is uneven, of course the temperature of the module on the front will be uneven. The radiator material is theoretically better to use copper, but due to price reasons, it is mostly aluminum alloy with high thermal conductivity. Another aspect is the cooling of the fan. The wind of the fan is large, and the uniformity of heat dissipation will increase, but the noise will also increase greatly. For example, the noise of the AB instrument is very high. How to improve the heat dissipation uniformity of the radiator is also a science.

5, the material and shape of the module

From a uniformity point of view, the module is best made of silver, followed by copper, and then aluminum alloy. The modules generally used in the market are aluminum or silver. The main reason is that the volumetric specific heat capacity of copper is too large, which is not conducive to the rate of temperature rise and fall. Silver has the same problem, so the silver modules on the market are actually hollow. But when it is made hollow, it has a negative effect on temperature uniformity. Therefore, the temperature uniformity of silver module instruments on the market is not higher than that of aluminum alloy, but the temperature rise and fall rate is higher. Because silver is easy to oxidize, some silver modules are gold-plated (don't think it is pure gold or a whole piece of gold or silver), and some are gray after surface oxidation treatment.

The aluminum alloy module also has shape problems. The large volume and the more connections between the holes are good for uniformity, but not good for the temperature rise and fall rate. Various manufacturers have also worked hard on the shape of the module, which is a balance.

6, temperature rise and fall speed

The temperature rise and fall speed is another important indicator of the PCR instrument. Too high a temperature rise and fall speed will also affect the uniformity of the temperature. Because the temperature rise and fall speed is high, the module does not quickly transfer heat, and the temperature difference caused by the difference of the semiconductor chips and the heat transfer will be greater.

In addition, what users want is the temperature in the tube when using the PCR instrument, and what the instrument can measure is the temperature of the sensor under the module. The temperature in the tube calculated by the results of theory and experiment is different from the actual temperature. The faster the temperature rise and fall. , The greater the difference.

In short, in addition to the six factors mentioned before, there are other influencing factors, such as the temperature drift of the sensor, the aging of the temperature processing circuit, and the uneven dust of the radiator. Some factors can be reduced or eliminated through regular calibration and maintenance.

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The difference between rapid PCR technology and rapid PCR instrument

The time required to complete a PCR reaction on the PCR machine depends on the following factors:

1. Module heating and cooling time

2. The temperature equilibration time between the module and the PCR tube

3. Extended time

4. Number of cycles

Let's do a little analysis on the above points, so that everyone can understand the difference between fast PCR technology and fast PCR instrument.

1. Module heating and cooling time

PCR machines generally indicate the heating rate and cooling rate, but most of the targets are the maximum temperature change rate, that is to say, the speed reached in an instant. The average temperature rise and fall speed related to the experiment is only indicated by a very small number of manufacturers. Calculated with an average of 2 degrees and 4 degrees (very few instruments can achieve an average temperature rise and fall of 4 degrees), the reduction from 95 degrees to 55 degrees is 20 seconds and 10 seconds, respectively, with a difference of 20 seconds for each cycle. Calculated with 35 cycles in general, it affects the experiment time of 700 seconds, which is less than 12 minutes. In fact, the overshoot problem must be considered, and the impact time will be less.

2. The temperature equilibration time between the module and the PCR tube

After the module temperature is reached, the temperature in the PCR tube is far from reaching (the thermal conductivity of plastic and water is more than 300 times worse than that of the aluminum block), and it takes time to balance the two. Generally, there are four ways to reduce this time. One is to overshoot the temperature of the module and drop to the target temperature when the temperature in the tube is about to reach, but too high an overshoot may cause the actual temperature to overshoot and affect the PCR reaction results; Reduce the volume of the PCR reaction solution, but when the volume becomes smaller, the reliability of the PCR reaction will be reduced; the third is to reduce the thickness of the PCR tube, but the strength is not enough when it is too thin; the fourth is to increase the degree of attachment of the PCR tube to the module.

3. Extended time

The extension time is related to the length of the PCR product and the synthesis speed of the DNA polymerase. If the length of the PCR product is less than 100bp, you may consider changing the extension time to 0 (ie, two-step method); using high-speed DNA polymerase is also a common method, but not all PCR experiments can use high-speed DNA polymerase.

4. Number of cycles

The number of cycles is related to the amount of template and testing requirements.

The so-called fast PCR technology is the integration of fast PCR instrument with ultra-thin tube (or special attachment technology) and high-speed DNA polymerase technology, which can complete the PCR reaction in a short time. When some manufacturers sell PCR machines to users, they do not explain the fast PCR technology, but only emphasize the high speed of the PCR machines (mostly, HuYou users use the maximum temperature rise and fall speed). It seems that after buying a fast PCR machine, the original 1.5-hour amplification time will be shortened to half an hour. However, after purchasing a fast PCR machine, the user realized that it was not so. There are many users who adopt the fast mode and cannot produce good experimental results at all. Especially for users who perform multiplex PCR amplification, such as reagents used by public security systems for identification.

Fast PCR technology and fast PCR instrument are two different things. The fast PCR instrument can only shorten the PCR reaction time by about 10 minutes at most. To achieve a truly fast PCR reaction, some other efforts are needed. Most users do not need rapid PCR technology at all.

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How to choose the right PCR function when facing different samples

As we all know, the PCR instrument, also known as the thermal cycler, is a special temperature-variable instrument that cooperates with the PCR process. The thermal cover is a part of the upper part of the instrument to prevent the sample from evaporating and condensing on the tube cover in a high-temperature environment to reduce the risk of reaction sample volume.

 PCR, also known as polymerase chain reaction, is a method to amplify target DNA fragments in vitro and is widely used in many aspects. It is mainly divided into three steps: high temperature denaturation, low temperature annealing, and temperature extension. Add primers, templates, Taq enzymes, dNTPs, buffers and other reactants to mix, and the double-stranded DNA hydrogen bond breaks and unwinds into a single strand at about 95°C. At a temperature of about 60°C, the single-stranded primers are combined with artificially designed primers according to the principle of base complementary pairing; and the temperature is raised to about 72°C, and a new semi-reserved copy complementary to the template DNA strand is synthesized by Taq enzyme extension chain.

During this process, the temperature of a certain section will be close to or higher than 100℃, and some samples will evaporate upwards with the high temperature. When encountering a PCR tube cover with a lower temperature, it will condense on the tube cover, and the sample in the tube will have a certain amount Loss, a small part does not participate in the cycle, resulting in a decrease in sample yield.

Prior to this, the instrument did not have this device. This problem was solved by adding paraffin oil to the sample, but in fact, the subsequent treatment of paraffin oil is more cumbersome. The emergence of subsequent hot lids can effectively avoid this risk. The temperature of the hot lid is generally 5-10°C higher than that of the module, so that the temperature of the upper end of the PCR tube in the module is always greater than the temperature of the reaction liquid. Will not evaporate upwards, so the volume of all sample reaction liquids will not change.

However, due to the different heights of the sample tubes, the non-adjustable thermal lid of the early instruments can only be used for sample tubes of uniform height. When using a shorter sample tube, an aluminum block needs to be added, which is more troublesome; the current adjustable thermal lid is divided into Knob type and self-adjusting type, etc. For some instruments, the height and pressure of the knob-type hot lid can be adjusted according to the sample tube, but you need to turn the knob back to the original position before changing the sample. In addition, there is a problem that the pressure is not well controlled. Too tight will denature the sample tube, etc. If it is too loose, it will not be able to achieve the corresponding temperature effect. Self-adjusting can adjust the height according to the height of the sample tube.

The so-called hot start is the step of adding Taq enzyme to the sample tube for temperature extension. However, Taq enzyme can also play a role in the room temperature environment. Therefore, it is necessary before adding reactants or denaturing at high temperature. Prevent the Taq enzyme from starting to work at an inappropriate temperature to avoid non-specific products in this situation. A certain number of templates and enzymes produce non-specific products, which means that the content of specific products will be reduced. Therefore, samples will be added on ice, or the PCR machine will be preheated first, and then quickly put into the PCR tube.

You can also mix the primer, Mg2+, dNTP, and buffer with the solidified and cooled wax and isolate the upper and lower layers of the remaining reaction components. In the denaturation stage, the wax is heated and melted, all ingredients are mixed, and the liquid wax floats to the upper part of the sample, acting as a thermal cover. But the subsequent wax treatment is more troublesome. The use of chemical modification or preparation of antigen-antibody complexes allows the enzyme activity to be controlled according to the temperature.

In the face of high-throughput samples, manual hot-start protection will be very time-consuming. Therefore, the PCR machine will set the temperature of the hot lid. Before the hot cover is heated to a certain temperature, the temperature of the module remains unchanged. When the temperature of the hot cover reaches the set value, the module heats up rapidly to achieve the effect of hot start.

In addition, many PCR machines have two options for heating mode: Tube and Block. This is because the temperature sensor of the instrument is in the lower part of the module, and the temperature in the sample tube cannot actually be measured. Under normal circumstances (Block), when entering the set temperature and running time, it means that the module will continue to reach the specified temperature for a corresponding time, and the temperature of the sample tube cannot quickly reach the specified temperature; while in Tube mode, the module temperature is first set If the temperature is higher than the set temperature, wait for the temperature of the sample tube to reach the specified temperature, and then lower the temperature of the module to the specified temperature, so that the temperature in the tube can reach the set value quickly. Different reaction volumes and requirements can use different modes: normal PCR or Tube mode for larger volume, Block mode can be used when holding for a long time and requiring higher temperature overshoot or smaller volume.

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Common questions and answers of the PCR machine

Common questions and answers of the PCR machine

1. Very low cDNA yield

possible reason:

*RNA template quality is low

*The mRNA concentration is overestimated

*There is a reverse transcriptase inhibitor or insufficient amount of reverse transcriptase in the reaction system

*Isotope Phosphorus 32 expired

*The reaction volume is too large, should not exceed 50μl

2. The amplified product has no band or very shallow band during electrophoresis analysis

*The most common reason is that your reaction system is PCR reaction system instead of RT-PCR reaction system

*Related to the total amount and purity of RNA at the beginning of the reaction

*It is recommended to add control RNA to the experiment

*When performing PCR amplification, the content of the reaction product of the first strand in the total reaction system should not exceed 1/10

*It is recommended to use Oligo (dT) or random primers instead of gene-specific primers (GSP) for first-strand synthesis. Due to the secondary structure of the RNA template, such as circular results, it is possible that GSP cannot anneal to the template; or SSII reverse transcriptase cannot effectively extend from this primer.

*The target mRNA contains a strong transcription stop site, you can try the following methods to solve it:

a. Increase the reaction temperature of the first chain to 50°C.

b. Use random hexamers instead of Oligo (dT) for the first chain reaction.

3.Produce non-specific bands

*Use RT negative control to detect whether it is contaminated with genomic DNA. If the PCR result of the RT negative control also shows the same band, the sample needs to be reprocessed with DNase I.

*In a PCR reaction, non-specific initial amplification will lead to non-specific results. Annealing at a temperature lower than the primer Tm 2 to 5 ℃, reducing the amount of magnesium ions or target DNA will reduce the generation of non-specific results.

*Due to the different methods of mRNA shearing, the selection of primers will lead to different RT-PCR results.

4. Produce smear bands

*The content of the first strand product in the PCR reaction system is too high

*Reduce the amount of primers

*Optimize PCR reaction conditions/reduce PCR cycles

*When DNase is used to treat RNA samples contaminated with DNA, the oligonucleotide fragments produced will produce non-specific amplification, which will generally appear as a diffuse background.

5. Produce large-molecular-weight diffuse bands

*In most cases, it is caused by non-specific initiation and extension caused by too low annealing temperature

*For long fragment PCR, it is recommended to dilute the cDNA concentration in the reaction system to 1:10 (or 1:100-1:200)

6. In the absence of reverse transcriptase, control RNA to obtain amplification results

*Usually caused by trace amounts of DNA in the control RNA. Because it is impossible to eliminate all DNA templates during in vitro transcription. It is recommended that the first strand cDNA be diluted 1:10, 1:100, 1:1000 times to eliminate the effects of DNA contamination.

*It may be a band of primer dimer

7.Amplification product stays in the sample well

*It is possible that the amount of template is too high and the PCR result produces a high molecular weight DNA jelly. It is recommended to dilute the results of the first strand by at least 100 times before performing a second amplification.

*In addition, if the annealing temperature used in the second PCR is 5°C lower than the Tm value of the primer, the annealing temperature can be appropriately increased or a hot start can be performed to improve specificity.

8. What is the difference between SSⅢ and SSⅡ?

*With higher thermal stability (up to 50°C)

*Has a longer half-life (up to 220 minutes)

*No inhibition to PCR

*Dry ice transportation

*Tdt activity is lower

9. Why do some people prefer to use SSⅢ instead of ThermoScript?

If ThermoScript is not stored properly, it will cause a rapid decrease in activity, while SSⅢ is more stable.

10. Why use gene-specific primers (GSP)?

GSP is best when amplifying low-abundance transcripts. OligodT primers are recommended for reverse transcription of high-quality RNA and full-length transcripts; random primers are used for reverse transcription of mRNA fragments.

11. Under what circumstances need to use RNase H?

When RNA/DNA hybrids cannot be denatured normally in the first round of PCR

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Causes of abnormal PCR amplification bands and solutions

When using PCR, the most frequently encountered problem is that there will be differences in amplification. Take this opportunity to discuss with you the reasons and solutions for the out-of-time PCR amplification strips.

False negative, no amplified band

The key steps of PCR reaction are ① preparation of template nucleic acid; ② primer quality and specificity; ③ enzyme quality; ④ PCR cycle conditions. Finding the reasons should also be analyzed and researched for the above links.

Template: ①The template contains miscellaneous protein; ②The template contains Taq enzyme inhibitor; ③The protein in the template is not digested, especially the histone in the chromosome; ④The template is lost too much or phenol is inhaled when the template is extracted and prepared; ⑤ The template nucleic acid is not completely denatured. When the quality of the enzymes and primers is good, no amplified bands will appear. It is most likely that the sample is digested, and the template nucleic acid extraction process is faulty. Therefore, it is necessary to prepare an effective and stable digestion solution. The program should also be fixed and should not be changed at will .

Enzyme inactivation: It is necessary to replace with a new enzyme, or use both the old and the new enzyme at the same time to analyze whether the enzyme activity is lost or not enough to cause false negatives. It should be noted that sometimes forgot to add Taq enzyme or ethidium bromide.

Primers: The quality of primers, the concentration of primers, and whether the concentrations of the two primers are symmetrical are common reasons for PCR failure or unsatisfactory amplified bands and easy diffusion. The quality of the primer synthesis of some batches is problematic. One of the two primers has a high concentration and the other has a low concentration, resulting in low-efficiency asymmetric amplification. The countermeasures are: ①Select a good primer synthesis unit. ②The concentration of the primer depends not only on the OD value, but also pay attention to the primer stock solution for agarose gel electrophoresis. The primer band must appear, and the brightness of the two primer bands should be roughly the same. Strips, PCR may fail at this time, and it should be resolved through consultation with the primer synthesis unit. If one primer has high brightness and one has low brightness, balance its concentration when diluting the primer. ③Primers should be stored in small amounts at high concentration to prevent multiple freeze-thaw cycles or long-term storage in the refrigerator, which may cause deterioration and degradation of the primers. ④ The primer design is unreasonable, such as the length of the primer is not enough, the formation of dimer between the primers, etc.

Mg2+ concentration: Mg2+ ion concentration has a great influence on PCR amplification efficiency. Too high concentration can reduce the specificity of PCR amplification, while too low concentration will affect PCR amplification yield and even make PCR amplification fail without showing amplified bands.

Change of reaction volume: Usually the volume used for PCR amplification is 20ul, 30ul, 50ul or 100ul. The volume used for PCR amplification is set according to the different purposes of scientific research and clinical testing. After making a small volume such as 20ul, When making a large volume, the conditions must be modelled, otherwise it is easy to fail.

Physical reason: Denaturation is very important for PCR amplification. For example, if the denaturation temperature is low and the denaturation time is short, false negatives are very likely; if the annealing temperature is too low, it can cause non-specific amplification and reduce specific amplification efficiency. Annealing temperature Too high affects the combination of primer and template and reduces PCR amplification efficiency. Sometimes it is necessary to use a standard thermometer to check the denaturation, annealing and extension temperatures in the thermal cycler or the water bath. This is also one of the reasons for PCR failure.

Target sequence variation: If the target sequence is mutated or deleted, which affects the specific binding of the primer and the template, or the primer and template lose their complementary sequence due to the deletion of a certain segment of the target sequence, the PCR amplification will not be successful.

False positive

The PCR amplified bands appearing are consistent with the target target sequence bands, and sometimes the bands are more neat and brighter.

Inappropriate primer design: The selected amplified sequence has homology with the non-target amplified sequence. Therefore, the amplified PCR product is a non-targeted sequence during PCR amplification. If the target sequence is too short or the primer is too short, false positives are prone to occur. Need to redesign the primers.

Cross-contamination of the target sequence or amplification product: There are two reasons for this type of contamination: one is the cross-contamination of the entire genome or large fragments, leading to false positives. This false positive can be solved by the following method: the operation should be careful and gentle to prevent the target sequence from being sucked into the sample gun or spilled out of the centrifuge tube. Except for enzymes and substances that cannot withstand high temperatures, all reagents or equipment should be autoclaved. All centrifuge tubes and sample injection tips should be used once. When necessary, the reaction tube and reagents are irradiated with ultraviolet rays before adding the specimens to destroy the existing nucleic acids. The second is the contamination of small fragments of nucleic acid in the air. These small fragments are shorter than the target sequence but have a certain degree of homology. It can be spliced to each other, and after complementing the primers, PCR products can be amplified, which can lead to false positives. Nested PCR can be used to reduce or eliminate them.

Non-specific amplification band

The bands appearing after PCR amplification are inconsistent with the expected size, large or small, or both specific amplified bands and non-specific amplified bands appear at the same time. The reasons for the appearance of non-specific bands are as follows: First, the primer and the target sequence are not completely complementary, or the primer polymerizes to form a dimer. The second is that the Mg2+ ion concentration is too high, the annealing temperature is too low, and the number of PCR cycles is too high. The second is the quality and quantity of enzymes. Often, enzymes from some sources are prone to non-specific bands while enzymes from another source do not. Too many enzymes sometimes cause non-specific amplification. The countermeasures are: redesign the primers when necessary. Reduce the amount of enzyme or switch to another source of enzyme. Reduce the amount of primers, increase the amount of template appropriately, and reduce the number of cycles. Properly increase the annealing temperature or use the two-temperature point method (93°C denaturation, 65°C annealing and extension).

Flaky drag or smear tape appears

In PCR amplification, smear bands, flaky bands, or carpet-like bands sometimes appear. The reason is often due to the excessive amount of enzyme or poor quality of the enzyme, too high dNTP concentration, too high Mg2+ concentration, too low annealing temperature, and too many cycles. The countermeasures are: reduce the amount of enzymes, or exchange enzymes from another source. ②Reduce the concentration of dNTP. Appropriately reduce the Mg2+ concentration, increase the amount of template, and reduce the number of cycles.

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How to prevent experimental contamination in PCR experiments

When performing PCR operations, the operator should strictly abide by some operating procedures to minimize the possibility of PCR contamination or prevent the occurrence of contamination.

1. Divide the operation area: At present, ordinary PCR can not achieve single-person single-tube and complete closed-tube operation. However, whether it can achieve single-person single-tube or not, the experimental operation is required to be carried out in three different areas. Pre-processing and post-processing should be carried out in different isolation areas:

(1) Specimen processing area, including preparation of amplification templates.

(2) Amplification zone, including preparation of reaction solution and PCR amplification.

(3) Product analysis area, gel electrophoresis analysis, product photography and preparation of recombinant clones.

(4) There must be a certain degree of isolation in each work area, special operation equipment, and a certain directionality. Such as: specimen preparation → PCR amplification → product analysis → product processing.

Remember: Do not take the products and equipment in the product analysis area to the other two working areas.

2. Distributed reagents: The reagents needed for PCR amplification should be prepared and dispensed on the ultra-clean workbench or negative pressure workbench equipped with ultraviolet lamps. All pipettes and pipette tips must be fixed in it, and cannot be used to absorb amplified DNA and DNA from other sources:

(1) The PCR water should be high-pressure double distilled water.

(2) The primers and dNTPs are prepared with high-pressure double-distilled water in the area without PCR amplification products.

(3) The primers and dNTP should be stored separately, and the time should be marked when the aliquots are used to find the cause in case of contamination.

3. Precautions for experimental operation: Although most of the residual contamination of the amplified sequence is the cause of false positive reactions, cross-contamination between samples is also one of the reasons. Therefore, it is not only necessary to be cautious and conscientious in performing amplification reactions, but also to pay attention to all aspects of sample collection, extraction and amplification:

(1) Wear disposable gloves. If the reaction solution is accidentally splashed, replace the gloves immediately.

(2) Use disposable tips. It is strictly forbidden to mix them with the tips in the PCR product analysis room. Do not expose the tips to the air for a long time to avoid aerosol pollution.

(3) Avoid splashing the reaction liquid. To avoid this situation when opening the reaction tube, centrifuge a little before opening the cap to collect the liquid at the bottom of the tube. If accidentally spilled on the gloves or the table, you should immediately change the gloves and wipe the table with dilute acid.

(4) When working with multiple samples, prepare the reaction mixture by mixing the dNTP, buffer, primer and enzyme first, and then aliquot, which can reduce operations, avoid contamination, and increase the accuracy of the reaction.

(5) Finally, the reaction template is added, and the reaction tube is tightly closed after adding.

(6) Setting up negative and positive controls and blank controls during operation can verify the reliability of the PCR reaction and also assist in judging the credibility of the amplification system.

(7) Use replaceable or high-pressure processable sample injectors as much as possible. Since the sampler is most susceptible to contamination by product aerosols or sample DNA, it is best to use replaceable or high-pressure sample injectors. If there is no such special sampler, at least the sampler should be dedicated during the PCR operation and cannot be cross-used, especially the sampler used for PCR product analysis cannot be used in the other two areas.

(8) Repeat the experiment, verify the results, and draw conclusions carefully.

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Precautions for purchase of PCR instrument

The temperature rise and fall rate, the accuracy and range of temperature control, the sample volume and the hot lid function are undoubtedly the key parameters that users are most concerned about.

When purchasing a PCR instrument, you should pay attention to several key parameters:

1. Maximum (rising/falling) temperature rate: heating rate 4℃/s, cooling rate 3℃/s. (A key parameter that may affect the rate of the PCR reaction).

2. Temperature accuracy and uniformity: temperature accuracy ≤±0.2°C, temperature uniformity ≤±0.3°C (factors that may affect the accuracy of the PCR reaction).

3. Sample size: 96×0.2ml, 96×0.2ml+77×0.5ml (factors affecting the price of PCR instrument).

4. Hot lid function: Stepless adjustable hot lid that can be positioned at any angle, which can adapt to test tubes of different heights.

5. Others: such as power-off protection function, software function, display screen, etc.

So how to choose a satisfactory PCR?

First of all, when purchasing a basic PCR, the first consideration is to ensure the needs of the experiment, focusing on the parameters required to ensure that the experiment can be completed, such as temperature accuracy uniformity, temperature rise/fall rate and sample size.

Secondly, such as power failure protection function, software function, adjustable hot cover function, etc., can enable us to experiment with more efficient performance.

Third, price and after-sales service are also issues that we need to pay attention to when purchasing PCR. Good after-sales service will make us feel the guarantee of quality and service, and it will bring great convenience to our work when the instrument needs to be overhauled and maintained.

ZHENGZHOU MINGYI INSTRUMENT EQUIPMENT CO.,LTD's core technical personnel are the first batch of PCR technology research engineers in China. We offer 2 year warranty after sale, any further questions, just contact us.