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Complete PCR primer design and1
Primer design basis (99% problems can be solved)

1. Primer length: The textbook requires 15-30bp, usually about 20bp. The actual condition is better to be 18-24bp to ensure the specificity, but the longer the better, too long primer will also reduce the specificity, and reduce the yield.

2. Primer amplification span: 200-500bp is appropriate, and the fragment can be expanded to 10kb under specific conditions.

3. Primer base: The content of G+C should be 40-60%, too little G+C amplification effect is not good, too much G+C is easy to appear non-specific bands. ATGC is best distributed randomly, avoiding clusters of more than 5 purine or pyrimidine nucleotides. Multi-gc for the 5′ end and intermediate sequences to increase stability, avoid rich GC at the 3′ end, no GC for the last 3 bases, or no GC for 3 of the last 5 bases.

4. Avoid secondary structure in primers, and avoid complementation between two primers, especially complementation at the 3 ‘end, otherwise primer dimer will be formed and non-specific amplified bands will be generated.

5. The bases at the 3 ‘end of primers, especially the last and penultimate bases, should be strictly paired to avoid PCR failure due to unpaired terminal bases.

6. Primers have or can be added with appropriate cleavage sites, and the amplified target sequence should preferably have appropriate cleavage sites, which is very beneficial for cleavage analysis or molecular cloning.

7. Specificity of primers: primers should have no obvious homology with other sequences in the nucleic acid sequence database.

8. Learn to use software: PP5, Oligo6, DNAstar, Vector NTI, primer3 (This online design works best).

The above content can solve at least 99% of primer design problems.

Control the details of primer design

1. Primer length

General primer length is 18~30 bases. In general, the most important factor determining the annealing temperature of the primer is the length of the primer. The annealing temperature of primer is generally selected (Tm value -5℃), and some directly use Tm value. The following formulas can be used to roughly calculate the annealing temperature of primers.

When the length of primer is less than 20bp: [4(G+C)+2(A+T)]-5℃

When the length of primer is greater than 20bp: 62.3℃+0.41℃(%G-C)-500/length-5℃

In addition, many software can also be used to calculate the annealing temperature, the calculation principle will be different, so sometimes the calculated value may have a small gap. To optimize PCR reactions, the shortest primers that ensure annealing temperatures of not less than 54℃ are used for the best efficiency and specificity.

Overall, primer specificity increases by a factor of four for each additional nucleotide, so that the minimum primer length for most applications is 18 nucleotides. The upper limit of primer length is not very important, mainly related to reaction efficiency. Because of entropy, the longer the primer, the lower the rate at which it anneals to bind to the target DNA to form a stable double-stranded template for DNA polymerase to bind.

When using software to design primers, the length of primers can be determined by TM value in turn, especially for primers of fluorescence quantitative PCR, TM=60℃ or so should be controlled.

2.GC content

Generally, the content of G+C in primer sequences is 40%~60%, and GC content and Tm value of a pair of primers should be coordinated. If the primer has A serious GC or AT tendency, appropriate amount of A, T or G and C tail can be added to the 5 ‘end of the primer.

3. Annealing temperature

The annealing temperature should be 5℃ lower than the unchain temperature. If the number of primer bases is small, the annealing temperature can be increased appropriately, which can increase the specificity of PCR. If the number of bases is large, the annealing temperature can be reduced appropriately. The annealing temperature difference between a pair of primers of 4℃ ~ 6℃ will not affect the PCR yield, but ideally the annealing temperature of a pair of primers is the same, which can vary between 55℃ ~ 75℃.

4. Avoid the secondary structure area of the amplification template

It is best to avoid the secondary structure region of the template when selecting the amplified fragment. The stable secondary structure of the target fragment can be predicted and estimated by relevant computer software, which is helpful for template selection. Experimental results show that the expansion is often unsuccessful when the free energy (△G) of the region to be expanded is less than 58.6lkJ/mol.

5. Mismatch with target DNA

When the amplified target DNA sequence is large, a primer may bind to multiple parts of the target DNA, resulting in multiple bands appearing in the result. This time it is necessary to use the BLAST software testing, website: http://www.ncbi.nlm.nih.gov/BLAST/. Select Align two sequences (bl2seq).

Pasting primer sequences to zone 1 and target DNA sequences to zone 2 is interchangeable, and BLAST calculates complementary, antisense, and other possibilities, so users don’t need to notice whether both chains are sense chains. You can also enter the GI number if you know the GI number of the sequence in the database, so you don’t have to paste a large section of the sequence. Finally, click Align at 3 to see if the primer has multiple homologous sites in the target DNA.

6. Primer terminal

The 3 ‘end of the primer is where the extension begins, so it is important to prevent mismatches from starting there. The 3 ‘end should not be more than 3 consecutive G or C, because this will cause the primer to be mistakenly triggered in the G+C enrichment sequence region. The 3 ‘end cannot form any secondary structure, except in special PCR (AS-PCR) reactions, the 3′ end of the primer cannot be mismatched. For example, if the encoding region is amplified, the 3 ‘end of primer should not be terminated at the third position of codon, because the third position of codon is prone to degenerate, which will affect the specificity and efficiency of amplification. When using annexation primers, refer to the codon use table, pay attention to biological preference, do not use annexation primers at the 3′ end, and use a higher concentration of primers (1uM-3uM).

7. Secondary structure of primers

The primers themselves should not have complementary sequences, otherwise the primers themselves will fold into hairpin structures, and this secondary structure will affect the binding of primers and templates due to steric hindrance. If artificial judgment is used, the continuous complementary bases of primers themselves should not be greater than 3bp. There should be no complementarity between the two primers, especially the complementary overlap of the 3 ‘end should be avoided to prevent the formation of primer dimers. In general, there should be no more than 4 consecutive bases homology or complementarity between a pair of primers.

8. Add markers or loci

The 5 ‘end has little effect on amplification specificity and can therefore be modified without affecting amplification specificity. The modification of primer 5 ‘end included: adding enzyme restriction site; Labeled biotin, fluorescence, digoxin, Eu3+, etc. Introduce protein binding DNA sequences; Introducing mutation sites, inserting and missing mutation sequences and introducing promoter sequences, etc. The extra bases will more or less affect the efficiency of amplification and increase the chance of primer dimer formation, but some concessions must be made for the next step. Additional sequences that do not exist on the target sequence, such as restriction sites and promoter sequences, can be added to the 5′ end of the primer without affecting specificity. These sequences are not included in the calculation of primer Tm values, but should be tested for complementarity and internal secondary structure.

9. Subclones

Most of the time, PCR is only preliminary cloning, and then we need to subclone the target fragment into various vectors, so we need to design additional bases for the next operation in the PCR step.

Some sequences designed for subcloning are summarized below.
Restriction endonuclease restriction site was added

Adding enzyme restriction sites is the most commonly used method for subcloning PCR products. Generally, the cleavage site is six bases, in addition to the 5 ‘end of the cleavage site need to add 2 ~ 3 protective bases. However, the number of protective bases required by different enzymes is different. For example, SalⅠ requires no protective base, EcoRⅤ requires 1 protective base, NotⅠ requires 2 protective bases, and Hind Ⅲ requires 3 protective bases.

LIC adds the tail

The full name of LIC is Ligation-Independent cloning, a cloning method invented by Navogen specifically for its part of the pET vector. The pET carrier prepared by LIC method has non-complementary 12-15 base single strand sticky ends, which complement the corresponding sticky ends on the target insert fragment. For amplification purposes, the primer 5′ sequence of the inserted fragment should complement the LIC vector. The 3′→5′ extranect activity of T4 DNA polymerase can form a single strand sticky end on the inserted fragment after a short time. Because the product can only be formed from the mutual annealing of the prepared insert fragment and the vector, this method is very fast and efficient, and it is directed cloning.
Directed TA clone add tail
TA cloning was unable to target the fragment into a vector, so later Invitrogen introduced a vector that could target cloning, which contained four prominent base GTGGS at one end. Therefore, in the design of PCR primers, complementary sequences should be added accordingly, so that fragments can be “oriented”.

If you’re short on time, you can try direct synthesis, combining the gene with the vector, which is what we call ET gene synthesis in musecularists.

D. In-Fusion cloning method

No ligase required, no long reaction required. As long as a sequence at both ends of the linearized vector is introduced In the design of primers, then the PCR product and the linearized vector are added into the in-fusion enzyme solution containing BSA and placed at room temperature for half an hour, the transformation can be performed. This method is particularly suitable for large volume conversion.

10. Merge primer

Sometimes, only limited sequence information is known about primer design. For example, if only the amino acid sequence is known, the merging primer can be designed. A merger primer is a mixture of different sequences representing all the different base possibilities that encode a single amino acid. To increase specificity, you can refer to the codon use table to reduce annexation according to base use preferences of different organisms. Hypoxanthine can be paired with all the bases to reduce the annealing temperature of the primer. Do not use the annexed bases at the 3′ end of the primer because the annealing of the last 3 bases at the 3′ end is sufficient to initiate PCR at the wrong site. Higher primer concentrations (1μM to 3μM) are used because primers in many annexation mixtures are not specific to the target template.

PCR raw materials control

1. Primer quantity

The concentration of each primer is 0.1 ~ 1umol or 10 ~ 100pmol. It is better to produce the required result with the lowest amount of primer. High concentration of primer will cause mismatch and non-specific amplification, and increase the chance of forming dimers between primers.

2. Primer concentration

The concentration of primers affects the specificity. The optimal primer concentration is generally between 0.1 and 0.5μM. Higher primer concentrations lead to amplification of nonspecific products.

3. Annealing temperature of primer

Another important parameter for primers is the melting temperature (Tm). This is the temperature when 50% of the primers and complementary sequences are represented as double-stranded DNA molecules. Tm is required to set PCR annealing temperature. Ideally, the annealing temperature is low enough to ensure effective annealing of the primers with the target sequence, but high enough to reduce nonspecific binding. Reasonable annealing temperature from 55℃ to 70℃. Annealing temperature is generally set 5℃ lower than Tm of primer.

There are several formulas for setting Tm, which vary greatly depending on the formula used and the sequence of primers. Because most formulas provide an estimated Tm value, all annealing temperatures are only a starting point. Specificity can be improved by analyzing several reactions that progressively raise the annealing temperature. Start below the estimated Tm-5℃, and gradually increase the annealing temperature in an increment of 2℃. Higher annealing temperature will reduce the formation of primer dimers and non-specific products. For best results, the two primers should have approximate Tm values. If the Tm difference of primer pairs is more than 5℃, the primers will show a significant false start by using a lower annealing temperature in the cycle. If the two primers Tm are different, set the annealing temperature to 5℃ lower than the lowest Tm. Alternatively, to increase specificity, five cycles can be performed first at annealing temperatures designed for higher Tm, followed by the remaining cycles at annealing temperatures designed for lower Tm. This allows a partial copy of the destination template to be obtained under tight conditions.

4. Primer purity and stability

The standard purity of custom primers is adequate for most PCR applications. The removal of benzoyl and isobutylyl groups by desalting is minimal and therefore does not interfere with PCR. Some applications require purification to remove any non-full-length sequences in the synthesis process. These truncated sequences occur because the efficiency of DNA synthesis chemistry is not 100%. This is a circular process that uses repeated chemical reactions as each base is added to make DNA from 3′ to 5′. You can fail in either cycle. Longer primers, especially those greater than 50 bases, have a large proportion of truncated sequences and may require purification.

The yield of primers is affected by the efficiency of synthetic chemistry and purification method. Biopharmaceutical companies, such as Cytology and Shengong, all use a minimum OD unit to ensure the total output of oligonucleoside. Custom primers are shipped in dry powder form. It is best to redissolve the primers in TE so that the final concentration is 100μM. TE is better than deionized water because the pH of water is often acidic and will cause the hydrolysis of oligonucleosides.

The stability of primers depends on storage conditions. Dry powder and dissolved primers should be stored at -20℃. Primers dissolved in TE at concentrations greater than 10μM can be stably stored at -20℃ for 6 months, but can only be stored at room temperature (15℃ to 30℃) for less than 1 week. Dry powder primers can be stored at -20 C for at least 1 year and at room temperature (15 C to 30 C) for up to 2 months.

5. Enzymes and their concentrations

At present, the Taq DNA polymerase used is basically the gene engineering enzyme synthesized by coliform bacteria. The amount of enzyme needed to catalyze a typical PCR reaction is about 2.5U(refers to the total reaction volume of 100ul). If the concentration is too high, it can lead to non-specific amplification; if the concentration is too low, the amount of synthetic product will be reduced.

6. Quality and concentration of dNTP

The quality of dNTP is closely related to the concentration and the efficiency of PCR amplification. The dNTP powder is granular, and its variability loses its biological activity if it is improperly stored. dNTP solution is acidic, and should be used in high concentration, with 1M NaOH or 1M Tris.HCL buffer solution to adjust its PH to 7.0 ~ 7.5, small amount of sub-packaging, frozen storage at -20℃. Multiple freeze-thawing will degrade dNTP. In PCR reaction, dNTP should be 50 ~ 200umol/L. Especially, attention should be paid to the concentration of the four DNTPS should be equal (equal mole preparation). If the concentration of any one of them is different from the others (higher or lower), mismatch will be caused. Too low concentration will reduce the yield of PCR products. dNTP can combine with Mg2+ and reduce the concentration of free Mg2+.

7. Template (target gene) nucleic acid

The amount and purification degree of template nucleic acid is one of the key links for the success or failure of PCR. The traditional DNA purification methods usually use SDS and protease K to digest and dispose specimens. The main functions of SDS are: dissolve lipids and proteins on the cell membrane, thus destroying the cell membrane by dissolving membrane proteins, and dissociate nuclear proteins in the cell, SDS can also combine with proteins and precipitate; Protease K can hydrolyze and digest proteins, especially histones bound with DNA, and then use organic solvent phenol and chloroform to extract proteins and other cell components, and use ethanol or isopropyl alcohol to precipitate nucleic acid. The extracted nucleic acid can be used as a template for PCR reactions. For general clinical detection specimens, a quick and simple method can be used to dissolve cells, lysate pathogens, digest and remove proteins from chromosomes to free target genes, and directly used for PCR amplification. RNA template extraction usually uses guanidine isothiocyanate or protease K method to prevent RNase from degrading RNA.

8.Mg2+ concentration

Mg2+ has a significant effect on the specificity and yield of PCR amplification. In general PCR reaction, when the concentration of various dNTP is 200umol/L, the appropriate concentration of Mg2+ is 1.5 ~ 2.0mmol/L. Mg2+ concentration is too high, the reaction specificity decreases, non-specific amplification occurs, too low concentration will reduce the activity of Taq DNA polymerase, resulting in the reduction of reaction products.

Magnesium ions affect several aspects of PCR, such as DNA polymerase activity, which affects yield; Another example is primer annealing, which affects specificity. dNTP and template bind to magnesium ion, reducing the amount of free magnesium ion required for enzyme activity. The optimal magnesium ion concentration varies for different primer pairs and templates, but a typical PCR starting concentration with 200μM dNTP is 1.5mM (note: For real-time quantitative PCR, use 3 to 5mM magnesium ion solution with a fluorescent probe). Higher concentrations of free magnesium ions increase yield, but also increase nonspecific amplification and decrease fidelity. To determine the optimal concentration, magnesium ion titrations were performed in increments of 0.5mM from 1mM to 3mM. To reduce the dependence on magnesium ion optimization, Platinum Taq DNA polymerase can be used. Platinum Taq DNA polymerase is able to maintain function over a wider range of magnesium ion concentrations than Taq DNA polymerase and therefore requires less optimization.

9. Pcr-promoting additives

Optimization of annealing temperature, primer design, and magnesium ion concentration is sufficient for highly specific amplification of most templates; however, some templates, including those with high GC content, require additional measures. Additives that affect the melting temperature of DNA provide another way to improve product specificity and yield. Full denaturation of the template is required for best results.

In addition, the secondary structure prevents primer binding and enzyme extension.

PCR additives, including formamide, DMSO, glycerin, betaine, and PCRx Enhancer Solution, enhance amplification. Their possible mechanism is to reduce the melting temperature, thus aiding the annealing of primers and assisting DNA polymerase extension through the secondary structure region. PCRx Solution has other advantages. Minimal magnesium ion optimization is required when used with Platinum Taq DNA polymerase and Platinum Pfx DNA polymerase. Thus, the Platinum technique is combined with the additive to increase specificity while reducing the dependence of the third approach, magnesium ion optimization. For best results, the concentration of additives should be optimized, especially DMSO, formamide, and glycerol, which inhibit Taq DNA polymerase.

Complete PCR primer design and2 Foreasy Taq DNA Polymerase

 

10. Hot start

Hot start PCR is one of the most important methods to improve PCR specificity in addition to good primer design. Although the optimal elongation temperature of Taq DNA polymerase is 72℃, the polymerase remains active at room temperature. Thus, non-specific products are produced when the holding temperature is lower than the annealing temperature during the preparation of the PCR reaction and at the beginning of the thermal cycle. Once formed, these nonspecific products are effectively amplified. Hot-start PCR is particularly effective when the sites used for primer design are limited by the location of genetic elements, such as site-directed mutations, expression cloning, or construction and manipulation of genetic elements used for DNA engineering.

A common method to limit the activity of Taq DNA polymerase is to prepare PCR reaction solution on ice and place it in a preheated PCR apparatus. This method is simple and inexpensive, but it does not complete the activity of the enzyme and therefore does not completely eliminate the amplification of non-specific products.

Thermal priming delays DNA synthesis by inhibiting an essential component until the PCR apparatus reaches denaturation temperature. Most manual thermal initiation methods, including delayed addition of Taq DNA polymerase, are cumbersome, especially for high-throughput applications. Other thermal priming methods use a wax shield to enclose an essential component, including magnesium ions or enzymes, or to physically isolate reactive components, such as templates and buffers. During the thermal cycle, the various components are released and mixed together as the wax melts. Like the manual hot start method, the wax shield method is cumbersome and prone to contamination and is not suitable for high throughput applications.

Platinum DNA polymerase is convenient and efficient for automatic hot start PCR. Platinum Taq DNA polymerase consists of recombinant Taq DNA polymerase combined with monoclonal antibody against Taq DNA polymerase. The antibodies are formulated by PCR to inhibit enzyme activity during prolonged temperature holding. Taq DNA polymerase was released into the reaction during the 94℃ insulation of the denaturation step, restoring full polymerase activity. In contrast to chemically modified Taq DNA polymerase for thermal initiation, Platinum enzyme does not require prolonged insulation at 94℃ (10 to 15 minutes) to activate the polymerase. With PlatinumTaq DNA polymerase, 90% of Taq DNA polymerase activity was restored after 2 minutes at 94℃.

 Complete PCR primer design and3

Foreasy HS Taq DNA Polymerase

11. Nest-PCR

Successive rounds of amplification using nested primers can improve specificity and sensitivity. The first round is a standard amplification of 15 to 20 cycles. A small fraction of the initial amplification product was diluted 100 to 1000 times and added to the second round of amplification for 15 to 20 cycles. Alternatively, the initial amplified product can be sized by gel purification. A nested primer is used in the second round of amplification, which can bind to the target sequence inside the first primer. The use of nested PCR reduces the possibility of amplification of multiple target sites because there are few target sequences complementary to both sets of primers. The same total number of cycles (30 to 40) with the same primers amplified the nonspecific sites. Nested PCR increases the sensitivity of limited target sequences (e.g., rare mrnas) and improves the specificity of difficult PCRS (e.g. 5′ RACE).

12. Descending PCR

Descending PCR improves specificity by using tight annealing conditions for the first few cycles of PCR. The cycle starts at an annealing temperature approximately 5℃ higher than the estimated Tm, then each cycle is reduced by 1℃ to 2℃ until the annealing temperature is below Tm 5℃. Only the destination template with the highest homology will be amplified. These products continue to expand in subsequent cycles, crowding out the amplified nonspecific products. Descending PCR is useful for methods where the degree of homology between primer and target template is not known, such as AFLP DNA fingerprinting.

 

 

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Post time: May-09-2023