Section 1217 flesh Original Address: PCR primer design Golden Rule (conventional PCR and qPCR) Au

Section 1217 flesh Original Address: PCR primer design Golden Rule (conventional PCR and qPCR) Author: Angel Xiaoxiao have a date
6. To randomly distributed base.
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 trypsin lead to primer dimer band, and reduce the effective concentration leaving the primer PCR reaction can not be normal.
G value is double-stranded DNA to form the desired free energy, which reflects the relative stability trypsin 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 can be analyzed using software Oligo 6)
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. trypsin Experiments show that the area be extended free energy ( G ) less than 58.6l kJ / mol, the amplification is often not successful. If the area can not be avoided, with the 7-deaza-2 '- deoxy GTP substituted trypsin dGTP success of amplification is helpful.
In analyze where, Duplex Formation whether upstream primer, or the downstream primer between the upstream and downstream primers, The most stable 3 '-Dimer absolute value should be less than 4.5kcal / mol, The most stable Dimer overall absolute value should generally be less than the number of kcal / mol with PCR annealing temperature, several experiments I feel at the time PCR annealing temperature of 65 , The most stable Dimer overall 6.7kcal / mol no problem.
About DNA primer design primer length: 15-25 bases GC content: about 50% if the primer binding to AT-rich region, consider replacing several bases with LNA, less primer length thereof and to avoid primer secondary structure and 3 'ends of dimer. Since competing points trypsin primer and template and the probe and the target and between the inner points hybrid, inverted repeats, and the like will cause trigger probe primer binding efficiency trypsin of reduced, so we choose primer dimer G is negative, that: <10 kcal / mol not contiguous G / C saved primer probe generally follow the following principles: The forward and reverse primers saved - 20 degrees, at a concentration of 10mM or 10 working concentration. probe should be protected from light and stored at -70 degrees, preferably in a lyophilized trypsin powder state, the retention of liquids in general working concentration of about two weeks. 2. Enter the target sequence, with BLASTn in http://www.ncbi.nlm.nih.gov/blast 3. Check to compare polymorphic sequence alignments and possible errors in these areas to avoid primers trypsin and probes 4. be designed to avoid direct repeat region in the target sequence, the repeat region hybridize so easily obtained product was non-primer binding, reduce the amplification efficiency of DNA and to reduce sensitivity of the analysis. 6. When RT step, with (http://www.bioinfo.rpi.edu/applications/mfold/rna/form1.cgi) tool detection folded situation at a specific temperature of the target sequence, to avoid secondary structure height region, the lower region of the probe and that the primer binding efficiency 7. The possible use of amplification product 60-150bp, GC content of 60% or slightly less efficient to determine the denaturation, more highly efficient reaction. trypsin GC content highly prone to non-specific trypsin sequence of reactions, a short sequence trypsin is amplified gDNA amplification time is shortened to reduce the likelihood of contamination. Easily synthesized short sequences used for amplification of multi-standard curve. OligodT reverse transcription with the best design template amplicon located close to the 3 'region Quantitative PCR is divided into the following categories: trypsin direct binding fluorescent dye product 1.SYBR green - specifically bind to double-stranded, fluorescently labeled fluorescent signal release 1.Taqman double fluorescent probes labeled probe 2.molecular Beacon fluorescently labeled fluorescently labeled hybridization probes 3. Dual probe so that a different approach to design is slightly different, but the basic principles are similar: The following lists 18 principles: First, keep up the template primer closely followed not have a stable hairpin dimers or between primer and primer, primer trypsin again not cause DNA polymerization (ie mismatch) in other non-target sites. Around trypsin these few basic principles, primers were designed to consider many factors, such as the length of the primer (primer length), the product of length (product length), the sequence value Tm (melting temperature), ΔG values (internal stability), primer-dimer and hairpin (duplex formation and hairpin), error priming site (false priming site), primers and product GC content (composition), and sometimes also on the modified primers, such as increasing the restriction point, trypsin the introduction of mutations. Oligo software analysis using primers designed, for example, have the following points: 1. The length of the primer is generally 15-30bp, trypsin commonly used 18-27bp, but not more than 38, because for too long will lead to its extended temperature is higher than 74 , i.e., the optimum temperature of the enzyme Taq. 2. The primer 3 'end of the sequence than the 5' trypsin end important. The primer 3 'end base is generally not A (3' end of the nucleotide sequence is preferably G, C, CG, GC), as A in the initiation site of the error initiation efficiency is relatively high. Also between the primer 3 'complementary to, dimers or hairpin structure of the end of the PCR reaction may also lead to failure. 5 'end of the sequence of PCR little effect, so often used to introduce modification sites or markers. 3. The GC content of the primers is generally 40-60%, with 45-55% is appropriate. Some GC content of the template itself is low or high, resulting in a GC content of the primers is not within the above range, this time should be as far as possible on the GC content of the downstream primer and remain close to the Tm value (on the GC content of the downstream trypsin primer should not differ too large), to facilitate the choice of the annealing temperature. If the GC ratio is exceeded, the primer in the 5 'end trypsin to increase As or Ts; and if AT ratio is too high, also in the 5' end to increase Gs or Cs. But also that: the original widely believed PCR primers should have 50% of GC / AT ratio of view is not correct to human genomic DNA as a template, with 81% AT primers can produce a single, specific, long 250 bp , a product containing 70% AT. There is no need to go to the complexity of the product and calculate the melting temperature of the primers, GC / AT ratio of PCR primers should be equal to or higher trypsin than the template to be amplified by the GC / AT ratio. 4. primer template sequence corresponding to the Tm value is preferably about 72 . (Tm value of the curve is better to select the vicinity of 72 degrees, 5 'to 3' drop shape also helps primer to initiate trypsin the polymerization reaction), trypsin at least in 55 - 5. ΔG value between 80 (free energy) reflects the primer intensity and degree of binding to the template. Generally, ΔG values primer preferably sinusoidal shape, i.e., the 5 'end and a higher intermediate ΔG value, while the 3' end ΔG value is relatively low, and not more than 9 (ΔG is negative trypsin Here absolute value), so it will help to prevent the right to initiate the reaction and caused the error. 3 'end trypsin of the double-stranded trypsin ΔG is 0 ~ - When 2 kcal / mol, PCR yields almost trypsin one hundred percent, with the increase in the absolute trypsin value of the decreased production in the - 6 only 40%, to - 8 fewer than 20 %, while the - 10 close to zero. 6. Possible errors initiation sites determined by primer sequence composition of the template sequence of similarity, the similarity is high, the error rate of initiation, the error caused by the initiation rate not higher than 100 in general, and so can not guarantee non-desired product fake belt. But for a specific template sequence, to compare the efficiency of initiation should be combined in the correct site. If the two are very different, for example, lead to efficiency trypsin in the correct trypsin site of 450 or more, and in the efficiency of initiation sites for the 130 error, and hard to find other, more appropriate primer, then this pair of primers is also acceptable. 7. Frq curve Oligo6 introduced a new index, the probability of the size of the repeat sequences revealed the presence of fragments. When selecting primers, should use a relatively low value Frq clips. 8. The energy of primer dimer and hairpin structures generally do not exceed 4.5, or prone to primer-dimer band but also reduce primer concentrations resulting PCR reaction does not proceed properly, a parameter associated with the dimer is base distribution of the 3 'end trypsin of the continuous GGG or CCC cause errors lead. Dimer formation of the more stable the higher energy value, the more does not meet the requirements. Dimer same energy value as low as possible hairpin. trypsin While some have a hairpin loop, which ΔG is - self-complementary primer 3 kcal / mol can also get a good result, but if it's 3 'end trypsin hairpin loop occupy very troublesome trypsin time, that will lead to internal primers extension reaction, reducing the number of participating in a formal reaction primer. Of course, if the hairpin loop at the 5 'end of the reaction is not much affected. 9. In Equation (4 G / C + 2 A / T - 5) calculating the Tm value, i.e., the annealing temperature. Select a lower Tm value of the primer annealing temperature for the annealing temperature of the reaction. 4-6 difference seems to yield little effect on the PCR. Preferably, to ensure the Tm value for each primer matches, and in the range of 70-75 10. You know, a more important factor is the difference between the template and the stability of the smaller primer melting temperature. The smaller the difference, the higher the efficiency of PCR. Because the melting temperature of DNA also depends on its length, so some researchers trypsin like the design very long, rather than seeking it very stable primers. However, trypsin the primer is too long will be difficult to avoid the formation of dimers and self-complementary, therefore, generally, or not at all. If the product is equal to or less than the expected length of 500 bp, choose a short (16 ~ 18 mer) primer: If the product of a long 5 kb, then with 24 mer primers. Some people get 40 kb product was 20 ~ 23 mer primers. 11. DNA sequencing and PCR, preferably 5 'end stability (e.g. higher GC content), while the 3' end is not stable (e.g. AT content of more) primers, the structure of such primers can effectively eliminate false initiate the reaction. This is based on the internal stability of the primer from experience. The 3 'end of the low stability reasons primer in these reactions can play a good role in that, near or at the 3' target site with non-base paired bases formed on the end of the stability enough to trigger DNA synthesis, so it will not produce spurious products. Therefore, in order to efficiently initiate the reaction, the 5 'end and a central portion primers must also form a double stranded target DNA. In contrast, with a stable, GC-rich 3 'end of the oligonucleotide does not require all of its nucleotide sequence are paired with the target sequence, only by virtue of its 3' terminus of the target sequence of any site fixedly engaged can initiate the reaction, to produce non-specific products. In any case, the oligonucleotide 3 'last five nucleotides stability trypsin end is less than - 9 kcal / mol, and is usually a specific probe or primer. Oligonucleotide trypsin 3 'end