primer

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Primer refers to nucleotide Polymerization At the beginning, it stimulates the synthesis of a specific nucleotide sequence Of macromolecule , and reactant These molecules are linked in the form of hydrogen bonds and are called primers. Primers are usually two artificially synthesized segments oligonucleotides Sequence: one primer complements a DNA template chain at one end of the target region, and the other primer complements another DNA template chain at the other end of the target region. Its function is to serve as the starting point of nucleotide polymerization, nucleic acid polymerase A new nucleic acid chain can be synthesized from its 3 'end. In vitro artificially designed primers are widely used polymerase chain reaction , sequencing and probe synthesis.^[1]

Chinese name: primer
Foreign name: primer
Type: RNA primer DNA primer
primer length: 18-27 bp, no more than 38

GC content: Generally 40-60%
Tm value: At about 72 ℃
Role: DNA replication
See publications: Terminology of Biochemistry and Molecular Biology, Science Press
Time of publication: 2008^[4]

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Existing with living things in nature DNA replication Primer（ RNA primer ）And polymerase chain reaction (PCR) DNA Primers). Generally speaking, primers refer to DNA primers, hereinafter referred to as primers.

primer design

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primer design

Primers are two artificially synthesized segments oligonucleotides Sequence, one primer and one end of the target gene DNA The template chain is complementary, and the other primer is complementary to the other DNA template chain at the other end of the target gene. In PCR（ polymerase chain reaction ）In technology, a segment is known Target gene Of nucleotide sequence , synthesize primers according to this sequence, and use PCR amplification The target gene DNA is broken into single strand after thermal denaturation, and the primer is corresponding to the single strand Complementary sequence Combine, and then DNA Polymerase The extended product can also be combined with the primer.

PCR Primer Design The goal is to find a pair of suitable nucleotide Fragment so that it can be effectively amplified Template DNA Sequence. As mentioned above, the quality of primers is directly related to the Specificity And success or failure. It is impossible to have an all inclusive rule for primer design to ensure the success of PCR, but following some principles will help primer design.

primer design There are 3 basic principle : First, the sequence of primer and template should be closely complementary, and then avoid the formation of stable Dimer or Hairpin structure Again, the primer cannot initiate DNA polymerization at the non target site of the template (i.e Mismatch )。 To implement these three basic principles, many factors need to be considered, such as primer length length ）, product length, sequence Tm value, primer and template formation Double chain Of Internal stability (internal stability, reflected by ∆ G), forming Primer dimer (prime dimer) and hairpin structure (duplex and hairpin) Emergy , at the false priming site Initiating efficiency , primer and product GC Composition, etc. If necessary, the primer should be modified, such as adding Restriction endonuclease Loci, introduced mutations, etc.^[2]

Best Area

DNA sequence The conservative region of is determined by comparing similar sequences among species. stay NCBI Search the same gene of different species on the, and use sequence analysis software (such as DNAman ）In alignment, the same sequence of each gene is the conserved region of the gene.

length

The primer length is usually 18-27 bp, but should not be greater than 38, because too long will lead to its extension temperature greater than 74 ℃, which is not suitable for Taq DNA Polymerase React.

GC content

Primed GC The content is generally 40-60%, too high or too low is not conducive to initiating the reaction. The GC content of upstream and downstream primers cannot differ too much

Tm value

The Tm value of the template position sequence corresponding to the primer at about 72 ℃ can make Renaturation The conditions are optimal. There are many methods to calculate Tm value, for example, according to the formula Tm=4 (G+C)+2 (A+T), Oligo software uses Nearest neighbor method (the nearestneighbor method)

3′ End avoidance Codon 3rd place

If the coding region is amplified, the 3 'end of the primer should not end at the third position of the codon, because the third position of the codon is easy to occur Degeneracy , will affect the specificity and efficiency of amplification.

Select T

When the 3 'end of primer is mismatched, different bases Initiating efficiency There are great differences. When the end base is A, even in the case of mismatch, there can be the synthesis of the trigger chain. When the end chain is T, the trigger efficiency of mismatch is greatly reduced. The trigger efficiency of G and C mismatch is between A and T, so T is the best choice at the 3 'end.

Base random distribution

The primer sequence should not be in the template Similarity It is high, especially the sequence with high similarity at the 3 'end. Otherwise, it is easy to cause false priming. One way to reduce the similarity between primers and templates is to use four primers Base The distribution of is better to be random, without clustering purine Or poly pyrimidine The exists of. In particular, the 3 'end should not exceed three consecutive Gs or Cs, because this will cause the primer to be incorrectly triggered in the GC enrichment sequence region.

Self avoid complementation

The primer itself should not have complementary sequences, otherwise the primer itself will fold into Hairpin structure (Hairpin) refold the primer itself. such Secondary structure It will affect the refolding combination of primer and template due to spatial hindrance. The primer itself cannot have 4 consecutive primers Base Complementarity of.

There should also be no complementarity In particular, complementary overlap at 3 'end shall be avoided to prevent Primer dimer (Dimer and Cross dimer). There should be no complementation of 4 consecutive bases between primers. If the primer dimer and hairpin structure are unavoidable, the △ G value should not be too high (should be less than 4.5kcal/mol, ∆ G value refers to the DNA double strand formation required free energy , this value reflects the interior of the double chain structure Base pair Of relative stability^[3] ）Otherwise, it is easy to produce primer dimer band and reduce primer Effective concentration The PCR reaction cannot be carried out normally.

The middle G value at 5 'end should be higher than that at 3' end

△G Value refers to DNA Double chain Forming the required free energy , which reflects the relative stability of base pairs within the double chain structure. The greater the △ G value, the more stable the double chain. The 5 'end and middle △ G value should be relatively high, while the 3' end △ G value should be relatively low（ absolute value No more than 9) primers. The △ G value at the 3 'end of the primer is too high, which is easy to form a double stranded structure at the mismatch site and trigger DNA polymerization 。 (△ G values at different positions can be analyzed with Oligo 6 software)

5 'end can be modified

The 5 'end of the primer determines the length of the PCR product, which has little effect on the amplification specificity. Therefore, it can be modified without affecting the specificity of amplification. Primer 5 'end modification includes: adding Restriction site ； Labeled biotin, fluorescence digoxin , Eu3+, etc; introduce protein Binding DNA sequence; Introduction of point mutation Insertional mutation 、 Deletion mutation Sequence; introduce Promoter Sequence, etc.

The primer extension starts from the 3 'end and cannot be modified. The 3 'end cannot form any Secondary structure probably.

Single chain without secondary structure

The main reason why some primers are invalid is the influence of the secondary structure of the single strand of the amplified product. It is better to avoid the secondary structure region when selecting amplified fragments. Use relevant software (such as RNA Structure) can Forecast estimate mRNA The stable secondary structure of is conducive to the selection of formwork. The experiment shows that when the free energy (△ G °) of the region to be expanded is less than 58.6l kJ/mol, amplification is often unsuccessful. If this area cannot be avoided, use 7-deaza-2 '- deoxidation GTP replace dGTP It is helpful for the success of amplification.

free energy

∆ G value refers to the free energy required for the formation of DNA double strand, which reflects the relative stability of base pairs within the double strand structure. Primers with lower ∆ G value at 3 'end (absolute value shall not exceed 9) and relatively higher ∆ G value at 5' end and middle should be selected. The ∆ G value at the 3 'end of the primer is too high, which is easy to form a double stranded structure at the mismatch site and trigger DNA polymerization.

Specificity

primer design After completion, BLAST test shall be carried out. If it does not have complementarity , you can proceed to the next experiment.

It is worth mentioning that the primer design difficulty of various templates is different. Some templates are difficult in their own conditions, such as high or low GC content, which makes it impossible to find primers that are very suitable for various indicators; PCR used for cloning purposes has a low degree of freedom of primer design because the product sequence is relatively fixed. In this case, we can only take the second place and try to meet the conditions.

design requirement

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When making Real Time, it is used for SYBR Green I A pair of primers and primers of general PCR in the primer design The parameters required on are different. Requirements for primer design:

● Avoid duplication Base , especially G

● Tm=58-60 degrees.

●GC=30-75%.

● There shall not be more than 2 G or C in the last 5 alkali bases at 3 'end

● Forward primer The closer to the probe, the better, but not overlapping.

● PCR amplification Product length: the product size of primer should not be too large, generally between 80-250 bp; 80~150 bp is most suitable (can be extended to 300 bp).

● Primer annealing temperature High, generally above 60 degrees;

Special attention should be paid to avoid the existence of primer dimer and non-specific amplification.

and primer design It should be taken into account that the primer should not Genomic DNA The ability of pollution to affect, that is, primers should cross Exon It is better that the primer can span the exon junction region, so that it can be more effective without being affected by genomic DNA contamination.

do dyestuff The most important thing is to find suitable primers and prevent pollution. For primers, you should be mentally prepared to select one or two usable primers from a large number of primers - it is very difficult to find suitable primers.

The function of BLAST should be to find the primer you designed through comparison. Among all the species' gene sequences that have been found and published in GENEBANK, besides your target gene, is there any same sequence with other species or other sequences, such as the same sequence with the sequence other than your target sequence, It is possible to expand the products of other sequences Specificity It is very poor, so it cannot be used.

Design software

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■ Oligo 6

Oligo 6

Oligo 6 is the most widely used primer design software. In addition to the simple and fast design and analysis of various primers and probes, it also has many advanced functions that other similar software does not have:

● Given the sequence of one PCR primer, search and design the sequence of another primer;

● Design according to the preference of different species for MM Degenerate primer ；

● Right Annular type DNA fragments, design Reverse PCR Primer;

● Design Multiplex PCR Primer;

● Design probes for LCR reaction to detect whether a mutation occurs;

● Analyze and evaluate whether primers designed by other ways are reasonable;

● Homologous sequence search and Cognate region Primer design;

● Enhanced primer/probe search. During primer design, you can "Lock" each parameter, such as Tm value Range and stability of primer 3 'end;

● Store results in various forms; support Multi user , each user can save their own special settings.

■ Primer Premier five

Primer Premier 5.0

Primer Premier 5.0 is an application software used to help researchers design the most suitable primers. Using its advanced primer search primer database, nested primer design, primer editing and analysis and other functions, you can design ideal primers with efficient amplification ability and primer sequences for amplification of PCR products up to 50kb.

The software is mainly edited by GeneTank sequence, Primer primer design , align sequence comparison, enzyme digestion analysis and motif analysis.

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