Basic concepts of PCR Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments in vitro. The main process of PCR technology is that a small single-stranded DNA fragment synthesized by humans (called a primer) specifically binds to a specific region of the template DNA, and then uses four dNTPs as substrates to form along the primer and template by DNA polymerase. The 3 'end of the double-stranded part is polymerized to form a DNA fragment to achieve the process of DNA amplification in vitro. Among them, the most important is the thermally stable DNA polymerase. Since the enzyme can also be stable at high temperatures above 97 degrees, we can process the DNA in the temperature range of 94 to 97 degrees to form a single strand, and then cool down (according to the primer Different and different, generally 50-55 degrees) until the appropriate amount of primers bound to the template. Finally, the temperature was raised to 72 degrees, and the polymerase polymerized to form double-stranded DNA. Classical method PCR system components A DNA template contains DNA fragments that need to be amplified. Two primers determine the starting and ending positions that need to be amplified. DNA polymerase (polymerase), copy the area to be amplified. Deoxyribonucleoside triphosphate (dNTP) refers to four kinds of deoxyribonucleic acids used to construct new complementary strands. The buffer system provides a chemical environment suitable for the function of the polymerase. In order to maintain the concentration of each component, H2O is diluted with water in the system Note: Some brand buffers do not contain Mg2 +, so you need to add the corresponding concentration of Mg2 +. Please add enzymes last, and water first. PCR steps 1. DNA denaturation (90 ℃ -96 ℃): Double-stranded DNA template under the action of heat, hydrogen bond breaks to form single-stranded DNA. 2. Annealing (25 ℃ -65 ℃): The temperature of the system is reduced, and the primers are combined with the DNA template to form local double strands. 3. Extension (70 ℃ -75 ℃): Under the action of Taq enzyme (about 72 ℃, with the best activity), dNTP is used as the raw material to extend from the 5 ′ end → 3 ′ end of the primer to synthesize the DNA strand complementary to the template. PCR cycle parameters 1. Initial Denaturation Complete denaturation of template DNA and complete activation of PCR enzymes are critical to the success of PCR. It is recommended that the heating time refer to the reagent instructions. Generally, the activation time of unmodified Taq enzyme is two minutes. 2. Denaturation steps in the cycle Generally, 95 ℃ for 30 seconds in the cycle is enough to completely denature various target DNA sequences. If possible, the step time can be shortened. If the denaturation time is too long, the enzyme activity will be damaged. If the target sequence is too short, the denaturation of the target sequence will not be complete, which may cause amplification failure. 3. Primer annealing The annealing temperature needs to be determined from various aspects. Generally, the Tm value of the primer is used as a reference, and the annealing temperature is appropriately adjusted down according to the length of the amplification. Then make an estimate based on this experiment. Annealing temperature has a greater influence on the specificity of PCR. 4. Primer extension Primer extension is generally carried out at 72 ℃ (Taq enzyme optimal temperature). However, when the amplification length is short and the annealing temperature is high, this step can be omitted. The extension time depends on the length of the amplified fragment. It is generally recommended to be above 1000 bp, and the derivative of Pfu and its derivatives is set to 1 min / kbp . 5. Number of cycles Most PCRs contain 25-40 cycles, and too much is prone to non-specific amplification. 6. Last extension After the last cycle, the reaction was maintained at 72 ° C for 5-15 minutes. The primer is extended completely, and the single-stranded product is annealed to double-stranded. Result analysis 1. False negatives, no amplified bands The key steps of the PCR reaction are: ①preparation of template nucleic acid, ② quality and specificity of primers, ③ enzyme quality and ④ PCR cycling conditions. Looking for the cause should also be analyzed and studied for the above links. Template: ①The template contains miscellaneous proteins, ② The template contains Taq enzyme inhibitors, ③ The protein in the template is not digested and removed, especially the histones in the chromosome, ④ Too much is lost when extracting the template, or inhaled phenol. ⑤ The template nucleic acid is not completely denatured. When the quality of enzymes and primers is good, there is no amplification band. It is most likely that the sample is digested. The template nucleic acid extraction process has a problem. Therefore, to prepare an effective and stable digestion treatment solution, the procedure should also be fixed and should not be changed at will. . Enzyme inactivation: new enzymes need to be replaced, or both old and new enzymes are used at the same time to analyze whether false negatives are caused by the loss or lack of enzyme activity. It should be noted that sometimes forget to add Taq enzyme or ethidium bromide. Primers: The quality of the primers, the concentration of the primers, and whether the concentrations of the two primers are symmetrical are common reasons for PCR failure or unsatisfactory amplification bands and easy dispersion. Some batches have a problem with the quality of primer synthesis. Two primers have a high concentration and a low concentration, resulting in asymmetric amplification with low efficiency. The countermeasures are: ①Select a good primer synthesis unit. ②The concentration of primers depends not only on the OD value, but also on the primer stock solution for agarose gel electrophoresis. There must be primer bands, and the brightness of the two primer bands should be roughly the same, such as one primer has a band, one primer has no The band, which may fail in PCR at this time, should be resolved through consultation with the primer synthesis unit. If one primer has high brightness and one has low brightness, the concentration should be balanced when diluting the primer. ③ Primers should be stored in small quantities in high concentrations to prevent multiple freeze-thaw cycles or long-term storage in the refrigerator to cause the primers to deteriorate and fail. ④ Primer design is unreasonable, such as primer length is not enough, dimer formed between primers, etc. Mg2 + concentration: Mg2 + ion concentration has a great influence on PCR amplification efficiency. Too high a concentration can reduce the specificity of PCR amplification. Too low a concentration can affect the yield of PCR amplification and even fail PCR amplification without bands. Change in reaction volume: Usually the volume used for PCR amplification is 20ul, 30ul, 50ul. Or 100ul, how much volume should be used for PCR amplification, is set according to different purposes of scientific research and clinical testing. After making a small volume such as 20ul, and then making a large volume, it must be a mold condition, otherwise it will easily fail. Physical reason: Denaturation is very important for PCR amplification. If the denaturation temperature is low and the denaturation time is short, false negatives are most likely to occur; if the annealing temperature is too low, it may cause non-specific amplification and reduce the specific amplification efficiency. Annealing temperature is too high Highly affects the binding of primers to templates and reduces the efficiency of PCR amplification. Sometimes it is necessary to use a standard thermometer to detect the denaturation, annealing and elongation temperatures in the thermal cycler or water bath, which 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 to the template, or the primer and template lose the complementary sequence due to a certain deletion of the target sequence, PCR amplification will not be successful. 2. False positives The bands of PCR amplification appearing are consistent with the bands of the target sequence of interest, sometimes the bands are more tidy and the brightness is higher. Primer design is not suitable: the selected amplification sequence has homology with the non-target amplification sequence, so when performing PCR amplification, the amplified PCR product is a non-target sequence. Target sequences that are too short or primers that are too short are prone to false positives. Need to redesign primers. Cross-contamination of target sequences or amplification products: There are two reasons for this contamination: One is cross-contamination of the entire genome or large fragments, resulting in false positives. This kind of false positive can be solved by the following methods: the operation should be careful and gentle to prevent the target sequence from being sucked into the sampling gun or splashing out of the centrifuge tube. Except for enzymes and substances that cannot withstand high temperatures, all reagents or equipment should be autoclaved. The centrifuge tubes and sample feed tips used should be used once. If necessary, before adding the specimen, the reaction tube and reagents are irradiated with ultraviolet rays to destroy the existing nucleic acids. The second is the nucleic acid contamination of small fragments in the air. These small fragments are shorter than the target sequence, but have a certain degree of homology. Can be spliced ​​with each other, after complementary to the primer, the PCR product can be amplified, which leads to the generation of false positives, which can be alleviated or eliminated by nested PCR. 3. A non-specific amplification band appears The bands that appear after PCR amplification are not consistent with the expected size, either larger or smaller, or both specific and non-specific amplification bands appear. The reason for the appearance of non-specific bands: First, the primer is not completely complementary to the target sequence, 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 too many PCR cycles. The second is the quality and quantity of enzymes. Some sources of enzymes are prone to non-specific bands and the other sources of enzymes do not appear. Excessive amounts of enzymes sometimes cause non-specific amplification. The countermeasures are: redesign primers if necessary. Reduce the amount of enzyme or replace the enzyme from another source. Reduce the amount of primers, increase the amount of template appropriately, and reduce the number of cycles. Appropriately increase the annealing temperature or use the two-temperature point method (93 ℃ denaturation, annealing and extension around 65 ℃). 4. A flake or smear appears PCR amplification sometimes appears smearing bands or sheet bands or carpet-like bands. The reason is often due to too much enzyme quantity or poor enzyme quality, dNTP concentration is too high, Mg2 + concentration is too high, annealing temperature is too low, too many cycles caused. The countermeasures are: reduce the amount of enzyme, or exchange the enzyme from another source. ② Reduce the concentration of dNTP. Appropriately reduce the Mg2 + concentration. Increase the amount of templates and reduce the number of cycles. Q & A Q1. In what order should the PCR components be added? A. Water-buffer-dNTP or Mg + -enzyme, aliquot, template. Q 2. How to design a controlled experiment? A. PCR generally requires two controls: Positive control: Use a system that is easier to amplify. For example, the system that can be expanded from the previous time can also use 16s, because this is the best expansion. Negative control: that is, your target system for this amplification does not need to add Taq enzyme. Q 3. Does the PCR product need to be purified by gel? A. If the amplified product of gel analysis has only one band, gel purification is not required. If other miscellaneous bands can be seen, it may be a dimer that has accumulated a large number of primers. A small number of primer-dimer moles are also very high, which results in a high proportion of clones with primer-dimer, rather than the target insert. For this purpose, gel purification is required before cloning. Q 4. Is the higher the template concentration, the better? A. The template concentration is not as high as possible, sometimes it is counterproductive Recommended Products Common PCR instrument Quantitative PCR instrument Electrophoresis equipment PCR primer PCR reagent PCR control Specific PCR kit PCR cloning kit RNA RNase detection / removal RT-PCR reagent RT-PCR standard quantitative PCR reagents Quantitative PCR labeling PCR cloning vector Pipette (Pipette) PCR tube PCR strip PCR plate real-time quantitative PCR capillary other The above is the novice experimental guide of PCR technology organized by editors. Dining Chair Modern,Dining Chair Velvet,Dining Chair Modern Elegant,Dining Chair Wood Bosa Furniture Co.,Ltd. , https://www.bosafurniture.com