Selection of oligonucleotide primers
1. Introduction The selection of oligonucleotide primers is usually the key to the success of the entire amplification reaction. The selected primer sequence will determine the size and position of the PCR product, and the Tm value of the amplified region, which is an important physical parameter related to the yield of the amplified product. Good primer design can avoid the background and non-specific products, and can recognize cDNA or genomic templates even in RNA-PCR. Primer design also greatly affects the amplification yield: if a crudely designed primer is used, there will be little or no product; and the amount of product obtained using a properly designed primer can be close to the theoretical output value during the exponential phase of the reaction. Of course, even with good primers, it is still necessary to optimize the reaction conditions, such as adjusting the Mg2 + concentration, using special co-solvents such as dimethyl sulfoxide, formamide and glycerol.
Computer-aided primer design is more effective than manual design or random selection. Some factors that affect the effect of primers in the PCR reaction, such as dissolution temperature, possible homology between primers, etc., are easily encoded and defined in computer software. The high speed of the computer can complete a large number of calculations on the primer position, length, and other possibility of primer conversion to adapt to the user's special conditions. By testing thousands of combinations and adjusting various parameters, primers suitable for users' special experiments can be proposed. Therefore, the overall "quality" of the primers selected by the computer software (set by the user in the program parameters) is guaranteed to be superior to the primers derived manually.
It should be noted that the primer does not have to be completely homologous to the template, so it can contain various modifications of the promoter sequence, restriction enzyme recognition site or 5 'end. This modification of the primer will not hinder the PCR reaction, but will be used in the future Amplicons play a role.
2. Basic PCR primer design parameters The purpose of primer design is to strike a balance between two targets: amplification specificity and amplification efficiency. Specificity refers to the frequency of errors. Primers with poor or inferior specificity will produce extra irrelevant and unwanted PCR amplicons, which can be seen on EB stained agarose gel; primer efficiency refers to the product of a pair of primers amplified in each PCR cycle How close it is to theoretically double the amount of growth.
â‘ Primer length;
Specificity is generally controlled by primer length and annealing temperature. If the annealing temperature of the PCR is set within a few degrees close to the primer Tm value (the melting temperature of the primer / template duplex), 18 to 24 base oligonucleotide chains have good sequence specificity of. The longer the primer, the less template is triggered during amplification annealing. In order to optimize the PCR reaction, the shortest primer that ensures the dissolution temperature is not lower than 54 ° C can obtain the best efficiency and specificity.
In general, it is best to seek safety within the range allowed by specificity. Each additional nucleotide increases the specificity of the primer by a factor of four; in this way, the shortest primer length for most applications is 18 nucleotides. It is still wise to design the primers so that the synthetic oligonucleotide chain (18-24 polymer) is suitable for a variety of experimental conditions.
②Secondary structure of primers includes primer dimer, hairpin structure, inter-primer dimer, etc. These factors will affect the binding of primers and templates and thus affect the efficiency of primers. For the dimer formed at the 3 'end of the primer, the ΔG should be controlled to be greater than -5.0kcal / mol or less than three consecutive base complements, because the primer dimer in this case may further form a more stable structure. The requirements for the middle or 5 'end of the primer can be appropriately relaxed. The hairpin structure formed by the primer itself also has a greater influence on the primer-template binding at the 3 'end or near the 3' end; the factors that affect the stability of the hairpin structure are in addition to the bond energy of the complementary base pairing and the stem-loop structure There is also a great relationship. Try to avoid primers with a hairpin structure at the 3 'end.
③ Primer GC content and Tm value PCR primers should maintain a reasonable GC content. The Tm value of a 20-base oligonucleotide chain containing 50% G + C is approximately in the range of 56-62 ° C, which can provide sufficient heat for effective annealing. The GC content and Tm value of a pair of primers should be coordinated. Primer pairs with poor coordination have poor efficiency and specificity because reducing the Tm value results in loss of specificity. In this case, the higher the Tm value of the primer, the greater the chance of error. If the annealing temperature is too high, primer pairs with low Tm values ​​may not work at all. When selecting a new pair of primers from a batch of oligonucleotides that have been synthesized within a specific sequence, this coordination of GC content and Tm value is critical. In general, the Tm value of a pair of primers should not exceed 2 to 3 degrees Celsius as much as possible. At the same time, the Tm value of the primer and product should not differ too much, preferably within 20 degrees Celsius.
â‘£Additional primer sequence and annealing temperature If additional sequence information needs to be added to the primer, such as T7RNA polymerase binding site, restriction site or GC hairpin structure can use longer primers. Generally speaking, the addition of extraneous sequences at the 5 'end of the primer will not affect the annealing of the primer-specific sequence. Sometimes, a large number of bases that are not paired with the template are added to the primer, and 4 to 5 amplification cycles can be performed at a lower annealing temperature; then, assuming that the sequence at the 5 'end of the primer has been added to the template, the calculation The remaining cycles are performed at the annealing temperature.
An important consideration when adding restriction enzyme sites to primers is that effective cleavage of most restriction enzymes requires 2 to 3 non-specific extra bases at the 5 'end of their recognition sequence, which increases the primer's non-specific The length of the template-specific sequence. Another disadvantage of the long primer sequence is that it affects the precise calculation of the dissolution temperature, which is necessary to determine the annealing temperature during the PCR reaction. For primers less than 20 bases, the Tm value can be calculated based on Tm = 4 (G + C) +2 (A + T). For longer primers, the Tm value needs to consider the kinetic parameters and be obtained from the calculation method of "nearest neighbor". Most existing PCR primer design software adopts this method.
⑤The 3 'end nucleotides of the primer make up the primer 3' end and the base pair of the template are very important for obtaining good results, and the mismatch of the last 5 to 6 nucleotides at the 3 'end of the primer should be as much as possible Less. If there is too much mismatch at the 3 'end, reducing the annealing temperature of the reaction to compensate for this mismatch will have no effect, and the reaction is almost doomed to failure. Another problem with the 3 'end of the primer is to prevent homology within a pair of primers. Special care should be taken that the primers are not complementary, especially at the 3 'end. The complementarity between primers will lead to the appearance of unwanted primer duplexes, and the PCR product obtained in this way is actually the amplification of the primer itself. This will create a competitive PCR state between the primer duplex product and the natural template, thereby affecting the success of the amplification. The stability of the 3 'end of the primer is determined by the base composition of the 3' end of the primer, and the ΔG of the 5 bases at the end is generally considered. The size of this value has a greater impact on amplification. If the negative value is large, the 3 'end is highly stable, the amplification efficiency is higher, and it is also more prone to ectopic initiation. It should be noted that the 3 'end of the primer should avoid T as much as possible. Experiments show that primers ending in T can be effectively extended even if they mismatch with T, G or C.
â‘¥ PCR product length and position within the target sequence All computer programs provide selection of PCR product length range. Generally speaking, the length of the PCR product has an effect on the amplification efficiency. In specific applications, the length of the PCR product depends in part on the template material.
The specific length of the expected product often depends on the needs of the application. If the goal is to establish a clinical test method for determining specific DNA fragments, a small DNA amplification product of 120 to 300 bp may be the best. The product should have good specificity and high production efficiency, and contain enough information that can be used for probe capture hybridization experiments. Products in this length range can be obtained by using a two-step amplification cycle method, thereby reducing amplification time.
Other PCR methods have different optimal product lengths. For example, when using quantitative RNA-PCR to detect gene expression, the product should be large enough to form a competitive template, so that the product and competitor can be easily distinguished on the gel. These products are generally in the range of 250-750 bp.
⑦Supplementary note: If you are looking for PCR primers within the cDNA sequence, you need to pay special attention to two points: First, try to keep the primers and products within the coding region of the mRNA, because this is a unique sequence for generating proteins, unlike the 3 'end non-coding region It is homologous to many other mRNAs; second, try to place primers on different exons in order to distinguish RNA-specific PCR products from those produced from contaminating DNA in size.
If the purpose of PCR is to clone a specific sequence of a gene or cDNA, the size of the product is pre-selected according to the specific application. Here, the computer program can provide information about the selected primer pair flanking the desired region.
When selecting primers to amplify DNA from different species, the 5 'and 3' end untranslated region sequences of the mRNA should be avoided because they may not have any homology.
3. Degenerate primer design â‘ When designing degenerate primers, be sure to check the degeneracy of the selected amino acid genetic code in the target amplification region. Obviously, we expect to choose the amino acid with the least degeneracy to achieve the purpose of improving specificity.
â‘¡ Pay full attention to the preference of the species for codons, and choose codons with high frequency of use to reduce the degeneracy of the primers.
â‘¢ Try to avoid degeneration of the 3 'end. For most amino acid residues, it means that the 3' end of the primer should not be located at the third position of the codon.
â‘£ Use deoxyhypoxanthine (dI) instead of degenerate bases in some polysemous positions.
4. Sequencing primer design Of course, the design of sequencing primers is generally done by sequencing companies, if you need to design yourself; then in addition to the general standards for primer design mentioned above, there are two points to note:
â‘ The standardization of the specificity of sequencing primers should be stricter, that is, the specificity should be given priority in design. Because in the sequencing reaction, if the primer and the template are annealed at an unexpected position and trigger chain extension, the result will greatly interfere with the result and even cause the result to be unreadable.
â‘¡ The Tm value of the sequencing primer is appropriately higher. Most sequencing reactions now use heat-resistant sequencing-grade DNA polymerase to catalyze, and use PCR thermal cycling program. The Tm value of the selected sequencing primer is slightly higher, which helps the reaction to smoothly cross the secondary structure region of the template to be tested, and also helps to reduce non-specific reactions.
5. The design of the probe The design of the probe has its own design characteristics according to different uses. Here is just a discussion of the general principles:
â‘ The length of the probe is generally between 20-50 nucleotides, too long synthesis cost is high, and prone to polymerase synthesis errors, long hybridization time. Too short will decrease the specificity.
â‘¡ Note that the content of G and C should be controlled at 40-60%, and at the same time, no more than 4 consecutive bases are repeated to avoid non-specific hybridization.
â‘¢The probe's own sequence cannot form a dimer, nor can there be a "hairpin" structure. This requirement is much stricter than the design of ordinary primers.
â‘£If the target of the probe is a mixture of multiple genes, the similarity between the probe and the unrelated genes must be controlled below 70%.
Analysis and countermeasures of common problems in PCR.
Electrophoresis detection time of PCR products It is generally believed that PCR products should be electrophoretically detected within 48 hours. Some are best detected by electrophoresis on the same day. After 48 hours, the band pattern will appear irregular or even disappear.
Trouble shooting guide
1. False negative, no amplification bands The key steps of PCR reaction are: â‘ preparation of template nucleic acid, â‘¡ quality and specificity of primers, â‘¢ enzyme quality, â‘£ 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 insufficiency of enzyme activity. It should be noted that sometimes forget to add Taq enzyme during PCR or ethidium bromide during electrophoresis.
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: â‘ Select a good primer synthesis unit. â‘¡The 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. â‘¢ The primers should be stored in small portions with high concentration during use, to prevent repeated freezing and thawing or long-term storage in the refrigerator to cause the primers to deteriorate and fail. â‘£ 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 of reaction volume: the volume used for PCR amplification is usually 20μl, 30μl, 50μl or 100μl. The volume used for PCR amplification is set according to different purposes of scientific research and clinical testing. After making a small volume such as 20μl, When making a large volume, you must re-examine the conditions, otherwise it is easy to fail.
Physical reason: Temperature 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 lead to non-specific amplification and reduce the specific amplification efficiency; if the annealing temperature is too high, it will affect the binding of the primer to the template and reduce PCR amplification. Increase efficiency. Sometimes it is necessary to use a standard thermometer to check the denaturation, annealing and extension temperature in the PCR instrument or water bath, which is also one of the reasons for the failure of PCR.
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 positive 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. Such false positives can be solved by the following methods: ①Care should be taken when handling to prevent the target sequence from being sucked into the sampler or splashing out of the centrifuge tube. ②All reagents or equipment should be autoclaved except for enzymes and other substances that are not resistant to high temperature. The centrifuge tubes and injection tips should be used once. ③ If necessary, before adding the specimen, the reaction tube and reagents are irradiated with ultraviolet rays to destroy the existing nucleic acid. 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 the nested PCR method.
3. There is a non-specific amplification band The band after PCR amplification is not consistent with the expected size, either larger or smaller, or a specific amplification band and a non-specific amplification band appear at the same time. 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. Secondly, the quality and quantity of enzymes. Some sources of enzymes are prone to non-specific bands and another source of enzymes does not appear. Excessive amounts of enzymes sometimes cause non-specific amplification. The countermeasures are: ①If necessary, redesign the primer. ② Reduce the amount of enzyme or exchange the enzyme from another source. ③ Reduce the amount of primers, increase the amount of template appropriately, and reduce the number of cycles. ④ Appropriately increase the annealing temperature or use the two-temperature point method (93 ℃ denaturation, annealing and extension around 65 ℃, also called two-step method).
4. Smearing or smearing occurs CR amplification sometimes occurs with smearing or sheeting or carpet-like bands. The reason is often due to the excessive amount of enzyme or the poor quality of the enzyme, the concentration of dNTP is too high, the concentration of Mg2 + is too high, the annealing temperature is too low, and the number of cycles is too large. The countermeasures are as follows: â‘ Reduce the amount of enzyme, or exchange the enzyme from another source. â‘¡ Reduce the concentration of dNTP. â‘¢ Appropriately reduce the Mg2 + concentration. â‘£ Increase the amount of template and reduce the number of cycles.
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