More disgusting than CCTV Duan was the informant and the original address: PCR primer design of the

More disgusting than CCTV Duan was the informant and the original address: PCR primer design of the 11 golden rules: Ivy
PCR primers were designed for 11 golden rules 1. The primer abdi ibrahim is preferably designed in the conservative region of cDNA template. DNA sequences of conserved regions between species by comparing the determined sequence of similarity. Search for the same gene in different species of NCBI, by sequence analysis software (eg DNAman) alignment (Alignment), the same sequence of each gene is conserved region of the gene. 2. The length of the primers is generally between 15 to 30 bases. Primer length (primer length) is commonly 18-27 bp, but not greater than 38, since too long will result in extending the temperature is greater than 74 , suitable for Taq DNA polymerase reaction. 3. Primer GC content between 40% ~ 60%, Tm value is preferably close to 72 . GC content (composition) is too high or too low is not conducive to initiate the reaction. GC content of the upstream and downstream primers can not be that much difference. In addition, abdi ibrahim the downstream primer Tm value (melting temperature) is an oligonucleotide melting temperature, i.e., under certain conditions of salt, 50% of the oligonucleotide duplex melting temperature. Effective start-up temperature, generally higher than the Tm value of 5 ~ 10 . If according to the formula Tm = 4 (G + C) +2 (A + T) estimated Tm value of the primer, the effective Tm of the primers 55 ~ 80 , which is preferably close to 72 Tm value so that optimal abdi ibrahim refolding abdi ibrahim conditions . 4. primer 3 'end to avoid the first three codons. Such as the coding region was amplified, the primer 3 'end abdi ibrahim do not terminate abdi ibrahim in the first three codons, because the first three codons prone degenerate, it will affect the specificity and efficiency of amplification. 5. primer 3 'end can not choose A, the best choice for T. Xo / bEUv primer 3 'end with the wrong time, the efficiency of initiation of different bases there are significant differences, when the bottom of the base as A, even in the case of mismatches, can also lead to synthetic chain, and When the bottom strand is T mismatch initiator efficiency is greatly reduced, G, C mismatches abdi ibrahim between initiation efficiency A, between T, so the 3 'end the best choice T. 6. To randomly abdi ibrahim distributed base. The primer sequences within the template should not have high similarity, particularly the 3 'end of the sequence similarity higher, otherwise abdi ibrahim easily lead to errors lead (False priming). Reduce primer template A method abdi ibrahim of similarity is that the distribution of the four bases in the primers are preferably random, do not have the presence of polypurine or polypyrimidine. In particular, the 3 'end should not be more than three consecutive G or C, because it would be in the GC-rich primer sequence region mis-priming. 7. There should be no primer complementary sequences between themselves and primers. Primers complementary sequence itself should not exist, otherwise the primer itself will be folded into a hairpin abdi ibrahim structure abdi ibrahim (Hairpin) primer annealing itself. This secondary structure due to steric hindrance affecting renaturation primer template binding. Primers complementary to itself can not have consecutive four bases. Nor should have complementarities between the two primers, in particular, should avoid overlapping complementary 3 'end to prevent primer dimers (Dimer and Cross dimer) formation. Can not have four consecutive complementary bases between the primers. Primer-dimer and hairpin if unavoidable, they should try to make it G value is not too high (less than 4.5kcal / mol). Otherwise easily lead to primer dimer band, and reduce the effective concentration leaving the primer PCR reaction can not be normal. 8. primers 5 'end and intermediate G values should be relatively high, and the 3' end of the G lower value. G value is double-stranded DNA to form the desired free energy, which reflects abdi ibrahim the relative stability of the duplex structure of the internal base pairs, G larger the value, the more stable duplex. Should be selected 5 'end and intermediate G value is relatively high, and the 3' end of the G lower value (absolute value is not more than 9) primers. Primer 3 ' G value side is too high, easily formed at a mismatch site double-stranded DNA structure and the polymerization initiator. ( G value of the different locations may be analyzed using software Oligo 6) 5 9. primer 'end can be modified, and the 3' end can not be modified. 5 'end of the primers determines the length of the PCR product, it is not specifically affect abdi ibrahim the amplification. Therefore, it can be modified without affecting the specificity of amplification. Primer 5 'end modifications include: add restriction sites; labeled abdi ibrahim biotin, fluorescent, digoxin, Eu3 +, etc; proteins binding DNA sequence is introduced; the introduction of point mutation, insertion mutation, deletion mutation sequence; introducing the promoter sequence and the like. Primer extension from the 3 'end of the beginning, can not make any modifications. 3 'end can not have any secondary structure formation is possible. 10. The single-stranded amplification product can not form secondary structures. Some primers to amplify the impact was mainly due to an invalid product of single-stranded secondary structure, selection amplified fragment is best to avoid regions of secondary structure. With related software (such RNAstructure) can predict the stability of mRNA secondary structure is estimated to help select a template. Experiments show that the area be extended free energy ( G ) less than 58.6l kJ / mol, the amplification is often not successful. If not avoid this area, with a 7-deaza-2'- deoxy-GTP replace abdi ibrahim dGTP amplification success is helpful. 11. The primer abdi ibrahim should have specificity. After R & {fN V = primer design is complete, and should be BLAST detection. If no other genes are complementary, you can make the next experiment. It is worth mentioning that the primer design abdi ibrahim templates for a variety of different difficulty. Some template itself difficult conditions, such as high or low GC content, leading to a variety of indicators are not found very suitable primers; used for PCR cloning purposes, because the product is relatively fixed sequence, primer design freedom of selection than low. In this case, only the next best thing, as far as possible to meet the conditions. When doing Real Time, a pair of primers and general primer abdi ibrahim PCR SYBR Green I method used when, in the primer design parameters required are different. Primer design requirements: 1) avoid duplication bases, particularly G. 2) Tm = 58-60 degrees. 3) GC = not have the 30 to 80%. 4) 3 'end of the last five bases more than two G or C. 5) The forward primer and the probe closer the better, but can not overlap. 6) PCR amplification product length: the product of the size of the primer is not too large, generally between 80-250bp can; 80 ~ 150 bp of the most appropriate (can be extended to 300 bp). 7) primer annealing temperature is high, generally above 60 degrees; pay special attention to avoid the presence of primer-dimers and nonspecific amplification. And primer design should take into account the primer must be free from pollution of genomic DNA of ability, that the primer should cross exon best primers across exon splice area, so you can more effectively from genomic DNA contamination impact. As for the design of software, PRIMER3, PRIMER5, PRIMER EXPRESS should be possible. Do dye method is to find the most critical to prevention efforts and make appropriate primers pollution. For a primer, you have to pick one or two primers can be mentally prepared for a lot of primers from --- to find a suitable primer is not easy. BLAST on the role should be by comparison and found that you designed this primer has been discovered and disclosed in the gene sequence is not GENEBANK species which, in addition to your target genes, and there are no other species or other sequence in which the presence of the same sequence as the target sequence and outside of your sequence identical sequence, it may expand the product abdi ibrahim to other sequences, abdi ibrahim then the specific primers on the poor, and thus can not be used.
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