Who can explain the process of PCR technology to me in detail?

Polymerase chain reaction (full name: polymerase chain reaction)

PCR for short. Polymerase chain reaction (PCR) is a method of enzymatic synthesis of specific DNA fragments in vitro, which consists of several reactions, such as high-temperature denaturation, low-temperature annealing (renaturation) and appropriate temperature extension. It is carried out in one cycle, so that the target DNA can be rapidly amplified, which has the characteristics of strong specificity, high sensitivity, simple operation and time saving. It can be used not only for basic research such as gene isolation, cloning and nucleic acid sequence analysis, but also for disease diagnosis or anywhere with DNA and RNA. Polymerase Chain Reaction (PCR) is also called cell-free molecular cloning or in vitro primer-directed enzymatic amplification of specific DNA sequences.

Edit the brief history of this paragraph.

Human beings have been studying nucleic acids for more than 100 years. In the late 1960s and early 1970s, people devoted themselves to the research of gene isolation technology in vitro. However, due to the low content of nucleic acid, the in vitro operation of DNA is limited to some extent. Khorana first put forward the idea of nucleic acid amplification in vitro in 197 1. But at that time, the method of gene sequence analysis was not mature, DNA polymerase with strong thermal stability had not been found, and the synthesis of oligonucleotide primers was still in the stage of manual and semi-automatic synthesis. This idea seems to have no practical significance. 1985, inspired by expressway, American scientist Kary Mullis invented PCR technology after two years' efforts, and published the first academic paper on PCR technology in Science magazine. Since then, PCR technology has been widely recognized by the life science community, and Kary Mullis won the Nobel Prize in chemistry from 65438 to 0993. However, the original PCR technology is quite immature, which is a complicated, costly and "useless" laboratory technology. At the beginning of 1988, Keohanog improved the authenticity of amplification by improving the enzyme used. Subsequently, Saiki and others extracted a thermostable DNA polymerase from the aquatic thermophilic bacteria living in the hot springs of Huangshi National Park, which greatly improved the amplification efficiency of PCR technology. It is precisely because of the discovery of this enzyme that PCR technology has been widely used and become the fundamental cornerstone of genetic and molecular biological analysis. In the following decades, the PCR method has been continuously improved: from qualitative analysis to quantitative determination; From the original gene that can only amplify a few kb, now it can amplify dozens of kb DNA fragments. Up to now, there are more than a dozen PCR techniques, for example, combining PCR with reverse transcriptase into reverse transcription PCR, and combining PCR with antibody into immune PCR.

Edit the technical principle of this paragraph.

Semi-conservative replication of DNA is an important way of biological evolution and passage. Double-stranded DNA can be denatured and melted into single strand under the action of various enzymes, and copied to the same bimolecular mussel with the participation of DNA polymerase and promoter according to the principle of base complementary pairing. In the polymerase chain reaction

In the experiment, it was found that DNA can be denatured and melted at high temperature, and can be refolded into double chains when it is cooled. Therefore, we can control the denaturation and renaturation of DNA by changing the temperature, design primers as promoters, and add DNA polymerase and dNTP to complete the replication of specific genes in vitro. However, DNA polymerase will be inactivated at high temperature, so it is necessary to add new DNA polymerase every cycle, which is not only cumbersome to operate, but also expensive, which restricts the application and development of PCR technology. The discovery of thermostable DNA polymerase-Taq enzyme is a milestone in PCR application. The enzyme can tolerate the high temperature above 90℃ without losing its activity, which makes the PCR technology very simple and greatly reduces the cost. PCR technology has been widely used and gradually applied in clinic.

Edit the working principle of this paragraph.

Similar to the natural replication process of DNA, its specificity depends on oligonucleotide primers complementary to both ends of the target sequence. PCR consists of three basic reaction steps: denaturation-annealing (renaturation)-extension: ① denaturation of template DNA: polymerase chain reaction when template DNA is heated to about 94℃.

After that, the double-stranded DNA of the template DNA or the double-stranded DNA formed by PCR amplification is dissociated, which can be combined with primers to prepare for the next round of reaction; (2) Annealing (renaturation) of the template DNA and the primer: after the template DNA is denatured into a single strand by heating, the temperature is reduced to about 40-60 DEG C, and the primer and the complementary sequence of the single strand of the template DNA are paired and combined; ③ Primer extension: DNA template-primer conjugate, under the action of DNA polymerase, uses dNTP as reaction raw material and target sequence as template, and according to the principles of base pairing and semi-conservative replication, synthesizes a new semi-conservative replication chain complementary to the template DNA chain, and repeats the three processes of cyclic denaturation-annealing-extension to obtain more "semi-conservative replication chains", which can be used as the template for the next cycle. It takes 2 ~ 4 minutes to complete a cycle, and the target gene to be amplified can be amplified by a million times in 2 ~ 3 hours.

Edit the reaction characteristics of this paragraph.

The specific determinants of highly specific PCR reaction are: ① the correct combination of the specificity of primers and template DNA; ② the principle of base pairing; ③ The authenticity of ③Taq DNA polymerase synthesis reaction; ④ The specificity and conservation of the target gene. The correct combination of primers and templates is the key. The combination of primer and template and the extension of primer chain follow the principle of base pairing. Because of the fidelity of polymerase synthesis reaction and the high temperature resistance of Taq DNA polymerase, the combination (renaturation) of template and primer in the reaction can be carried out at higher temperature, the specificity of combination is greatly increased, and the amplified target gene fragment can maintain high accuracy. By selecting highly specific and conservative target gene regions, its specificity will be higher. The amount of PCR products with high sensitivity increases exponentially, and the initial template to be tested can be amplified to the level of μg= 10-6 according to Pique (PG =10-2). A target cell can be detected from 6.5438+0 million cells; In virus detection, the sensitivity of PCR can reach 3 RFU (plaque forming unit); In bacteriology, the lowest detection rate is 3 bacteria. Simple and rapid PCR reaction uses high temperature-resistant Taq DNA polymerase. After the reaction solution is added at one time, denaturation-annealing-extension reaction is carried out in DNA amplification solution and water bath pot, and the amplification reaction is generally completed within 2 ~ 4 hours. Amplification products are generally analyzed by electrophoresis, and isotopes are not needed, so there is no radioactive pollution and it is easy to popularize. The purity of the specimen is low, and it is not necessary to separate viruses or bacteria from cultured cells. Crude DNA and RNA can be used as amplification templates. It can be directly used for DNA amplification and detection of clinical samples such as blood, body cavity fluid, mouthwash, hair, cells and living tissues.

Edit the five elements of this paragraph.

There are five main substances involved in PCR reaction, namely primers, enzymes, dNTP, templates and Mg2+ primers. Primers are the key to the specific reaction of PCR, and the specificity of PCR products depends on the complementary degree between primers and template DNA. Theoretically, as long as the sequence of any template DNA is known, complementary oligonucleotide chains can be designed as primers to amplify the template DNA in vitro by PCR. Primer design should follow the following principles: ① Primer length: 15-30bp, generally around 20bp. ② The primer amplification span: 200-500bp is appropriate, and the fragment of 10kb can be amplified under certain conditions. ③ Primer base: the content of G+C should be 40-60%. Too little G+C is not conducive to amplification, and too much G+C is easy to produce non-specific bands. ATGC should be randomly distributed to avoid the string arrangement of more than 5 purine or pyrimidine nucleotides. (4) Avoid the secondary structure in the primers, and avoid the complementarity between the two primers, especially the complementarity at the 3' end, otherwise primer dimer will be formed, resulting in non-specific amplification bands. ⑤ The bases at the 3' end of the primer, especially the last base and the penultimate base, should be strictly matched to avoid the failure of PCR caused by the mismatch of the terminal bases. ⑥ There is or can be a suitable restriction site in the primer, and it is better to have a suitable restriction site in the amplified target sequence, which is very beneficial to restriction analysis or molecular cloning. ⑦ Primer specificity: Primers should have no obvious homology with other sequences in the nucleic acid sequence database. Primer quantity: the concentration of each primer is 0. 1 ~ 1 umol or 10 ~ 100 pmol, and the lowest primer quantity is better to produce the required results. Higher primer concentration will lead to mismatch and nonspecific amplification, and increase the chance of dimer formation between primers.

Edit the reaction system and reaction conditions in this paragraph.

Standard PCR reaction system: 10× amplification buffer 10ul four dNTP mixtures, each containing 200 μm ol/L primer10 ~/00pmol template DNA 0. 1 ~ 2ug Taq DNA polymerase 2.5Mg2+. Five elements of PCR reaction: There are five main substances involved in PCR reaction, namely primer, enzyme, dNTP, template and buffer (Mg2+ is needed).

Select PCR reaction conditions to edit this paragraph.

The reaction conditions of PCR are temperature, time and cycle times. Setting of temperature and time: based on the principle of PCR, set three temperature points: denaturation-annealing-extension. In the standard reaction, the three-temperature point method is used. Double-stranded DNA denatures at 90 ~ 95℃, and then rapidly cools to 40 ~ 60℃. After annealing, the primer binds to the target sequence, and then it is quickly heated to 70 ~ 75℃. Under the action of Taq DNA polymerase, the primer chain extends along the template. For short target genes (when the length is 100 ~ 300 BP), the double temperature point method can be used, and annealing and extension temperature can be combined in addition to denaturation temperature. Generally, it is denatured at 94℃ and annealed at about 65℃ (at this temperature, Taq DNA enzyme still has high catalytic activity). ① denaturation temperature and time: low denaturation temperature and incomplete melting are the main reasons for PCR failure. Generally speaking, the time from 93℃ to 94℃ is enough to denature the template DNA. If it is lower than 93℃, the time needs to be extended, but the temperature should not be too high, because the high temperature environment has an impact on the activity of the enzyme. If the target gene template or PCR product cannot be completely denatured at this step, PCR will fail. ② Annealing (renaturation) temperature and time: Annealing temperature is an important factor affecting the specificity of PCR. After denaturation, the temperature is quickly reduced to 40℃ ~ 60℃, so that the primer can be combined with the template. Because template DNA is much more complicated than primers, the probability of collision and binding between primers and templates is much higher than that between complementary chains of templates. The annealing temperature and time depend on the length of primer, base composition and concentration, and the length of target sequence. For primers with 20 nucleotides and 50% G+C content, 55℃ is the ideal starting point for selecting the best annealing temperature. The renaturation temperature of primers can help to select the appropriate temperature by the following formula: Tm value (melting temperature) =4(G+C)+2(A+T) renaturation temperature =Tm value -(5 ~ 10℃). Choosing a higher renaturation temperature can greatly reduce the nonspecific binding between primers and templates and improve the specificity of PCR reaction. The renaturation time is generally 30 ~ 60 seconds, which is enough to completely combine the primer and the template. ③ extension temperature and time: the biological activity of Taq DNA polymerase is 70 ~ 80℃ 150 nucleotides/sec/enzyme molecule 70℃ 60 nucleotides/sec/enzyme molecule 55℃ 24 nucleotides/sec/enzyme molecule above 90℃, and DNA synthesis can hardly be carried out. The extension temperature of PCR reaction is generally 70 ~ 75℃, and the common temperature is 72℃. Too high extension temperature is not conducive to the combination of primers and templates. The time of PCR extension reaction depends on the length of the fragment to be amplified. Generally, for DNA fragments within 1Kb, the extension time of 1min is enough. The target sequence of 3 ~ 4 KB takes 3 ~ 4min；; The amplification of 10Kb needs to be extended to 15min. Excessive extension will lead to the appearance of non-specific amplification bands. For the amplification of low concentration template, the extension time is slightly longer.

Edit the enzyme and its concentration.

At present, there are two kinds of Taq DNA polymerase available, one is a natural enzyme purified from hydrothermal Bacillus, and the other is a genetic engineering enzyme synthesized by Escherichia coli. It takes about 2.5U of enzyme to catalyze a typical PCR reaction (when the total reaction volume is 100ul). Too high concentration will cause nonspecific amplification, and too low concentration will reduce the amount of synthetic products. The quality and concentration of dNTP are closely related to that of dNTP and the efficiency of PCR amplification. DNTP powder is granular, and it is easy to lose its biological activity if it is not stored properly. DNTP solution is acidic and should be prepared with 1M NaOH or 1M Tris after high concentration. Adjust the PH value of HCL buffer to 7.0 ~ 7.5, subpackage a small amount and freeze at -20℃. Repeated freezing and thawing will degrade dNTP. In the PCR reaction, dNTP should be 50 ~ 200 μm ol/L, especially the concentration of four dNTP should be equal (equimolar preparation). If any one of them is different from the others (higher or lower), it will cause mismatch. Too low concentration will reduce the yield of PCR products. DNTP can combine with Mg2+ and reduce the concentration of free Mg2+. Template (target gene) nucleic acid The quantity and purification degree of template nucleic acid is one of the key links in the success or failure of PCR. Traditional DNA purification methods usually use SDS and protease K to digest and treat samples. The main function of SDS is to dissolve the lipid and protein on the cell membrane, so as to dissolve the membrane protein, destroy the cell membrane and free the nuclear protein in the cell. SDS can also combine with protein for precipitation. Protease K can hydrolyze and digest protein, especially histones bound to DNA, then extract protein and other cell components with organic solvents phenol and chloroform, and precipitate nucleic acids with ethanol or isopropanol. The extracted nucleic acid can be used as a template for PCR reaction. In general clinical samples, a quick and simple method can be used to dissolve cells, crack pathogens and digest protein of chromosomes, so that the target gene can be freed and directly used for PCR amplification. The extraction of RNA template usually adopts guanidine isothiocyanate or protease K method to prevent RNase from degrading RNA. Mg2+ concentration has a significant effect on the specificity and yield of PCR amplification. In general PCR reaction, when the concentration of various dNTP is 200 μm ol/L, the appropriate concentration of Mg2+ is1.5 ~ 2.0 mmol/L. If the concentration of Mg2+is too high, the specificity of the reaction will be reduced and nonspecific amplification will occur. If the concentration is too low, the activity of Taq DNA polymerase will decrease, and the reaction products will also decrease.

Edit this task step.

Basic process of PCR reaction

The standard PCR process is divided into three steps (as shown): 1. DNA denaturation (90℃-96℃): Under the action of heat, the hydrogen bond of the double-stranded DNA template breaks to form single-stranded DNA; 2. Annealing (renaturation) (40℃-65℃): When the system temperature decreases, the primer and DNA template combine to form a local double strand. 3. Extension (68℃-75℃): Under the action of Taq enzyme (the best activity is about 72℃), dNTP is used as raw material to extend from the 5' end to the 3' end of the primer to synthesize a DNA strand complementary to the template. After denaturation, annealing and extension in each cycle, the DNA content doubles. At present, due to the short amplification region, some PCR products can be replicated in a short time even if the Taq enzyme activity is not optimal, so it can be changed to a two-step method, that is, annealing and extension are carried out at 60℃-65℃ at the same time, so as to reduce the heating and cooling process once and improve the reaction speed.

Edit the loop parameters of this paragraph.

1, initial denaturation. The complete denaturation of template DNA is very important for the success of PCR, and it is usually heated at 95℃ for 3-5 minutes. 2. The annealing temperature of primer annealing is generally determined by experiment (experience). Annealing temperature has a great influence on the specificity of PCR. 3. Primer extension Primer extension is generally carried out at 72℃ (the optimum temperature of Taq enzyme). The extension time depends on the length of the amplified fragment and the amplification efficiency of Taq enzyme used. 4. denaturation step in the cycle: generally, 95℃ for 30 seconds is enough to completely denature various target DNA sequences in the cycle: denaturation time is too long, which will destroy the activity of the enzyme, and when it is too short, the target sequence will not be completely denatured, which will easily lead to amplification failure. 5, the number of cycles Most PCR contains 25-35 cycles, which is easy to produce non-specific amplification. 6. Finally, after the last cycle, the reaction was maintained at 72℃ for 5- 15 minutes, so that the primer was completely extended and the single-stranded product was annealed into double-stranded. PCR-PCR frequently asked questions

Edit the electrophoresis detection time.

Generally, it is within 48 hours, and some of them are better than electrophoresis detection on the same day. After more than 48 hours, the band pattern is irregular or even disappears. The key links of false negative PCR reaction without amplification bands are ① preparation of template nucleic acid, ② quality and specificity of primers, ③ quality of enzyme and ④PCR cycle conditions. To find out the reason, we should also analyze and study the above links. Template: ① template contains protein, ② template contains Taq enzyme inhibitor, ③ protein in template has not been eliminated, especially histone in chromosome, ④ template is lost too much during extraction and preparation, or phenol is inhaled. ⑤ The template nucleic acid is not completely denatured. When the quality of enzyme and primer is good, there is no amplification band, which is probably due to the failure of sample digestion and template nucleic acid extraction process. Therefore, in order to prepare effective and stable digestive juice, the procedure should be fixed and cannot be changed at will. Inactivation of enzyme: it is necessary to replace the new enzyme or use the old and new enzymes at the same time, and analyze whether the false negative is caused by the loss or deficiency of enzyme activity. It should be noted that Taq enzyme or ethidium bromide is sometimes forgotten. Primers: The quality of primers, the concentration of primers and whether the concentrations of two primers are symmetrical are the common reasons for the failure of PCR or the unsatisfactory amplification band, which is easy to disperse. The quality of primer synthesis in some batches is problematic. One of the two primers has high concentration and the other has low concentration, which leads to low efficiency of asymmetric amplification. The countermeasures are as follows: ① The selected primers are used to synthesize a single position. ② The concentration of primer depends not only on OD value, but also on agarose gel electrophoresis of primer stock solution. There must be primer bands, and the brightness of the two primer bands should be roughly the same. For example, one primer has a band and the other primer has no band. At this time, PCR may fail, and it is necessary to negotiate with the primer synthesis unit. If one primer has high brightness and the other has low brightness, the concentration should be balanced when diluting the primer. (3) Primers should be stored in small packages with high concentration to avoid deterioration and degradation due to repeated freezing and thawing or long-term storage in the refrigerator. ④ The primer design is unreasonable. If the length of primers is not enough, dimers will be formed between primers. Mg2+ concentration: Mg2+ ion concentration has great influence on PCR amplification efficiency. Too high concentration will reduce the specificity of PCR amplification, and too low concentration will affect the yield of PCR amplification, and even make PCR amplification fail without producing amplification bands. Variation of reaction volume: Generally, the volumes used for PCR amplification are 20ul, 30ul and 50ul. Or 100ul, which volume to use for PCR amplification is set according to different purposes of scientific research and clinical testing. When making a small volume, such as 20ul, you must set the mode conditions, otherwise it is easy to fail.

Edit the physical reason for this paragraph.

Denaturation is very important for PCR amplification, such as low denaturation temperature and short denaturation time, which is prone to false negative; Too low annealing temperature will lead to non-specific amplification and reduce the efficiency of specific amplification. Too high annealing temperature will affect the combination of primers and templates and reduce the efficiency of PCR amplification. Sometimes it is necessary to use a standard thermometer to detect the denaturation, annealing and extension temperature of an amplifier or a water-soluble pot, which is also one of the reasons for the failure of PCR. Variation of target sequence: If the target sequence is mutated or deleted, which affects the specific binding between the primer and the template, or the primer and the template lose their complementary sequences due to the deletion of a certain segment of the target sequence, the PCR amplification will not succeed. False positive PCR amplification bands are consistent with the target sequence bands, and sometimes the bands are more neat and brighter. Improper primer design: the selected amplification sequence has homology with the non-target amplification sequence, so the amplified PCR product is the non-target sequence when PCR amplification is carried out. If the target sequence is too short or the primer is too short, it is easy to appear false positive. Primers need to be redesigned. Cross-contamination of target sequences or amplification products: There are two reasons for this contamination: First, cross-contamination of whole genome or large fragments leads to false positives. This 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 loading gun or splashed out of the centrifuge tube. Except for enzymes and substances that can't withstand high temperature, all reagents or equipment should be autoclaved. The centrifuge tube and injection gun head used should be used once. If necessary, before adding samples, the reaction tubes and reagents are irradiated with ultraviolet rays to destroy the existing nucleic acids. The second is the pollution of small fragments of nucleic acid in the air, which are shorter than the target sequence, but have certain homology. They can be spliced with each other, and after complementary with primers, PCR products can be amplified to produce false positives, which can be reduced or eliminated by nested PCR. Non-specific amplification bands appeared. The bands amplified by PCR are inconsistent with the expected size, large or small, or specific amplification bands and non-specific amplification bands appear at the same time. There are two reasons for the appearance of nonspecific bands: one is that the primers are not completely complementary to the target sequence, or the primers polymerize to form dimers. Secondly, too high Mg2+ ion concentration and too low annealing temperature are related to too many PCR cycles. Secondly, the quality and quantity of enzymes, often some enzymes from some sources are prone to non-specific bands while others are not, and sometimes non-specific amplification will occur when the amount of enzymes is too high. The countermeasure is: redesign the guide if necessary. Reduce the amount of enzyme or replace the enzyme from other sources. Reduce the amount of primers, appropriately increase the amount of templates, and reduce the number of cycles. Appropriately increase the annealing temperature or adopt the double temperature point method (denaturation at 93℃, annealing extension at about 65℃). PCR amplification has smear or smear band, and sometimes smear band or smear band or carpet-like band appears. The reasons are often too many enzymes or poor enzyme quality, too high dNTP concentration, too high Mg2+ concentration, too low annealing temperature and too many cycles. The countermeasure is: reduce the amount of enzyme, or change the enzyme from other sources. ② Reduce the concentration of dNTP. Reduce the concentration of Mg2+ appropriately. Increase the number of templates and reduce the number of cycles.

Edit the PCR product of this clone.

1) What are the best conditions for cloning PCR products? The best insertion fragment: vector ratio needs to be determined by experiments. 1: 1 (insert: vector) is often the best ratio, and the molar ratio of 1: 8 or 8: 1 is also acceptable. The ratio range should be determined. For ligation, 5ul 2X ligase, 50ng plasmid DNA, 1Weiss unit T4 ligase, and insert *** 10ul. Keep the temperature at room temperature 65438 0 hours, or stay overnight at 4℃. At these two temperatures, the carrier lacking T- projection will connect itself and produce blue dots. Keeping the temperature 1 hour at room temperature can meet most cloning requirements. In order to improve the connection efficiency, it is necessary to stay overnight at 4℃. 2) 2) Do PCR products need gel purification? For example, gel analysis has only one band, and gel purification is not needed. If other bands can be seen, it may be that the dimer volume is tired of a large number of primers. A small amount of primer dimer also has a high molar number, which will produce a high proportion of clones with primer dimer, rather than the target insert. So gel purification is needed before cloning. 3) If the target fragment is not recovered, what control experiment is needed? A) coating untransformed competent cells. If there is a colony, it means that ampicillin is ineffective, or the plasmid with ampicillin resistance is contaminated, or the colony with ampicillin resistance is produced. B) Transform the complete plasmid, calculate the colony growth number, and determine the transformation efficiency. For example, dilute 1ug/ul plasmid 1: 100, and transform 100ul competent cells with 1ul. After being diluted to 1000ul with SOC, spread 100ul on the board. After overnight culture, 1000 colonies were produced. The conversion rate is: the total number of colonies produced and the total number of DNA diffusion. The total amount of planking DNA is the amount used in the transformation reaction divided by the dilution multiple. Specifically, 10ng DNA was transformed, diluted to 1000u with SOC, containing 10 ng DNA, spread with110, and * * used 1 ng DNA. The transformation rate is: 1000 clones x 10 (cubic) ng/ planking 1 ng DNA ug = 10 (cubic) cfu/ ug transforming pGEM-T application 10 (cubic) cfu/ ug competence. C) pGEM-T positive control or PCR products were used to generate >: 20-40 blue spots (using the designated step10 (8th power) cfu/ ug competent cells), indicating that the vector lost T ... possibly ligase contaminated nuclease. T4 DNA ligase (M 180 1, M 1804, M 1794) has good quality standard and no nuclease pollution, so T4 DNA ligase from other sources should not be replaced. D) transforming high-frequency competent cells (10 (8th power) cfu/ug) with pGEM-T or pGEM-T Easy vector, and obtaining 100 colonies according to the specified experimental steps, of which 60% should be white spots, such as >: 20-40 blue spots, with no colonies or few colonies. 4) The result of the control experiment is good, but the target fragment is not recovered. What's wrong with the experiment A) The ligation can be maintained at room temperature for 65438 0 hours, which can satisfy most clones. In order to improve efficiency, it is necessary to stay overnight at 4℃. B) The inserted fragment is contaminated, resulting in deletion of 3'-T or inhibition of ligation and transformation. Therefore, the inserted fragment was mixed with pGEM-T positive control and then ligated. If the colony number of the control is reduced, the inserted fragment needs to be purified or re-prepared. If a large number of blue dots are produced, it means that the inserted fragment is contaminated by nuclease, which makes pGEM-T or pGEM-T easy to delete the vector 3'-T. C) The inserted fragment is not suitable for connection. Due to excessive ultraviolet irradiation, gel-purified inserts sometimes appear. Excessive ultraviolet radiation will produce pyrimidine dimer, which is not conducive to connection, and DNA must be purified again. D) There is no A at the end of the amplification product of heat-resistant DNA polymerase with repair function, which is needed for cloning pGEM-T or pGEM-T Easy vector. Adding Taq DNA polymerase and nucleotide can add one at the end. Please refer to the technical data of pGEM-T pGEM-T Easy carrier (TM042) for details. E) Highly repetitive sequences may be unstable, leading to deletion and rearrangement during amplification. If it is found that insertions are often deleted and rearranged, recombinant defective E.coli strains, such as SURE cells, should be used.

Edit the classification of this PCR reaction.

Overlapping PCR

Overlapping amplification gene splicing is an effective method for gene fusion and site-directed mutation based on common PCR technology. As we all know, the primer only needs to be effectively combined with the template, especially the 5'-end sequence does not need to be completely paired with the template, so one or even two restriction sites can be added to the 5'-end of the amplified primer for later cloning. The SOEing method takes advantage of this feature, mixing new sequences into two independent genes to achieve the purpose of overlapping regions between the two genes, and the combination of 3' ends makes gene fusion or site-directed mutation possible.

Reverse transcription polymerase chain reaction

RT-PCR is the abbreviation of reverse transcription PCR and real-time PCR. Reverse transcription -PCR (RT-PCR) is a widely used variant of polymerase chain reaction (PCR). In RT-PCR, RNA strands are reverse transcribed into complementary DNA, which is then used as a template for PCR amplification of DNA.