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Section 1217 flesh primer design has three basic principles: the sequence first primer and the template should be closely complementary, followed by avoiding the formation of stable biofisica dimers or hairpin structures between the primer and primer, primer again things not in the non-purpose-bit template DNA polymerization initiation point (ie mismatch). Specifically achieve this three basic principles need to consider many factors, such as the length of the primer (primer length), biofisica the product of the length (product biofisica length), Sequence Tm value (melting temperature), forming a duplex with the template primer of internal stability (internal stability, with G values reflect), primer-dimer formation (primer biofisica dimer) and hairpin (duplex formation and hairpin) energy value, triggering efficiency, primers and products GC content mismatch site (false biofisica priming site) (the composition), and so on. Need to be modified primers, if necessary, such as increasing the restriction enzyme sites, introduced mutations. biofisica According to the reference material and the author summarizes in practice, primer design should note the following points: 1. The length of the primer is typically 15-30 bp, commonly used is 18-27 bp, but not greater than 38, because for too long will lead to extending a temperature biofisica greater than 74 , suitable for Taq DNA polymerase reaction. 2. The primer sequences within the template should not have high similarity, especially 3 ' high sequence similarity ends, otherwise easily lead to mismatches. 3 'primer end appeared biofisica three or more consecutive bases, such as GGG or CCC, will also lead to increased risk of error. 3. The 3 'primer the bottom end of the base have a greater biofisica impact on the efficiency of Taq DNA synthesis enzymes. Bottom base in different positions cause different mismatched amplification efficiency, the bottom of the base for the A mismatch was significantly higher than the other three bases, and should be avoided in primers 3 ' end use of bases A. Further, primer dimers or hairpin structures can also cause PCR reaction to fail. 5' terminal sequence of the PCR modest impact, so often used to introduce modification sites or markers. biofisica 4. GC content of the primer sequence is generally 40-60%, 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. 5. Tm primer sequences corresponding template location biofisica value at about 72 enables optimal annealing conditions. Tm value is calculated for a variety of procedures, such as the formula Tm = 4 (G + C) +2 (A + T), a pair of primers consistent best use of the Oligo software is nearest neighbor (the nearest neighbor method) . 6. G value is double-stranded DNA to form the desired free energy, this value reflects the relative stability of the duplex structure of the internal base pairs. It should be the choice of 3 ' G lower end value (absolute value is not more than nine), and 5' End and intermediate G value is relatively high primer. 3 'primer G end of high value, easy to form a double-stranded structure in the mismatch site and cause DNA polymerization. 7. primer-dimer and hairpin energy value is too high (over 4.5kcal / mol) is easy to result in primer dimer band, and reduce the effective concentration leaving the primer PCR reaction can not be normal. 8. Modified primers generally in the 5' terminal increase restriction site, the sequence should be determined according to the respective next experiment to be inserted PCR product carrier. It is worth mentioning that the primer design templates for a variety biofisica 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 cloning purposes in the PCR product because of the 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. (Reprint) now for PCR primer design primer biofisica There are several: Oligo 6.22 Primer 5.0, etc. If there are no special requirements biofisica for primers can also be used to design primers biofisica generunner, biological pass http://www.ebiotrade.com/newsf/2006 -4 / 2006429175034.htm
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