PCR is widely used in genetic testing, paternity testing, blood screening, clinical diagnosis, and is also a very useful tool in molecular biology research because of its high specificity and reliability. However, PCR has some inherent shortcomings, which are mainly manifested in the fact that some non-specific amplification products will inevitably appear in PCR experiments. In order to overcome this shortcoming, researchers nowadays use hot start PCR to reduce the amplification of these non-specific products. Hot start PCR is a modified PCR method that has been shown to be a valuable tool for reducing the production of non-specific bands during target DNA amplification. There are many hot-start PCR methods, and a new hot-start PCR method has been introduced, in which deoxyribonucleic acid 5'-triphosphate (dNTP) is modified to produce heat-activatable derivatives of dNTP, which is then introduced into the 3'-terminal of the primer to produce a new heat-activatable primer, bringing new application prospects of the hot-start PCR method. The introduction of dNTP into the 3' end of the primer resulted in a new heat-activated primer, thus bringing new application prospects for hot-start PCR.
Principle
The basic principle of hot-start PCR experiments using heat-activated primers is to introduce a protective group into the primer, a key component of the PCR reaction system. Using conventional solid-phase synthesis techniques, two phosphotriester (PTE) modifying groups, 4-oxo-pentyl (OXP) and MAF, are easily introduced into the primer by a modified phosphoramidite reagent. phosphoramidite reagent. It has been shown that the OXP moiety has a promising application in hot-start PCR because the OXP protective group dissociates more readily from the primer at elevated temperatures, allowing the primer to quickly regain elongation activity, a transition that does not require additional routine processing.
This dissociation of the protective group may be caused by the acidification of the Tris base in the PCR reaction buffer, where heating causes the pH of the Tris base in the PCR buffer to decrease, e.g., the pH of the buffer is 8 at 25 ℃, but becomes 6 when the temperature reaches 95 ℃. Some researchers have investigated the dynamic effect of the conversion of PTE primers into PDE primers. In this study, PTE primers were co-incubated with PCR buffer (pH 8.4 at 25 °C) at 95 °C for about 40 min, and almost all OXP-primers were converted to PDE primers, with half of the conversion time being set at 8.5 min ± 1.5 min. The conversion of primers modified with two OXP moieties to the corresponding unmodified PDE primers is more complicated, and requires a two-step process. The removal of the OXP groups was accomplished in two steps. At 95℃, it takes 1~l.5 h to complete the transformation.
Operation method
Hot-start PCR experiments using heat-activated primers
Principle
The basic principle of hot-start PCR experiments using heat-activated primers is to introduce a protective group into the primer, a key component of the PCR reaction system. Using conventional solid-phase synthesis techniques, two phosphotriester (PTE) modifying groups, 4-oxo-pentyl (OXP) and MAF, are easily introduced into the primer by a modified phosphoramidite reagent. phosphoramidite reagent. It has been shown that the OXP moiety has a promising application in hot-start PCR because the OXP protective group dissociates more readily from the primer at elevated temperatures, allowing the primer to quickly regain elongation activity, a transition that does not require additional routine processing. This dissociation of the protective group may be caused by the acidification of the Tris base in the PCR reaction buffer, where heating causes the pH of the Tris base in the PCR buffer to decrease, e.g., the pH of the buffer is 8 at 25 ℃, but becomes 6 when the temperature reaches 95 ℃. Some researchers have investigated the dynamic effect of the conversion of PTE primers into PDE primers. In this study, PTE primers were co-incubated with PCR buffer (pH 8.4 at 25 °C) at 95 °C for about 40 min, and almost all OXP-primers were converted to PDE primers, with half of the conversion time being set at 8.5 min ± 1.5 min. The conversion of primers modified with two OXP moieties to the corresponding unmodified PDE primers is more complicated, and requires a two-step process. The removal of the OXP groups was accomplished in two steps. At 95℃, it takes 1~l.5 h to complete the transformation.
Materials and Instruments
Equipment: Move The basic procedure for hot-start PCR experiments using heat-activated primers can be divided into the following steps: A Synthesis and Dissolution of Heat-Activated Primers The primer design is the same as that of the normal PCR reaction, except that heat-resistant protective groups are introduced into the 3' end oligonucleotide bond or the sub-terminal bond of the primer during the synthesis process, which can be done in the company that synthesizes the corresponding primers. The synthesized primers can be dissolved in autoclaved purified water to a final concentration of 25-100 mmol/L as a storage solution. B. Preparation of PCR Reaction System PCR sample preparation is illustrated by the example of 20 μL and 50 μL PCR reaction systems (see Table 10-4). In a 200 μL microcentrifuge tube placed in an ice bath, add 0.2 mmol/L dNTP, 2.5 U Taq DNA polymerase, 1 X PCR buffer (depending on the supplier, the buffer should be appropriate, and usually contains Mgundefined), 0.5 μmol/L forward primer, 0.5 μmol/L reverse primer, and 1 μg to 1 μg of DNA template to the remaining volume. DNA template, and the remaining volume was made up by adding sterilized pure water. After mixing, centrifuge instantly to collect the reaction components at the bottom of the tube. C Reaction conditions ① Temperature of denaturation of starting template and primer deprotection group: 94 ℃, 10 min. ② Denaturation temperature: 94 ℃, 30 s. ③ Annealing temperature: 55 ℃, 30 s. ③ Annealing temperature: 55~60 °C, 30 s (Generally, the annealing temperature of 55~60 °C is more appropriate, and the primer will hybridize with the template for a certain period of time at this temperature.) ④ Intra-circular extension temperature: 72 ℃, 30 s (Generally, the extension time is I min/1 kb of base, and 30 s for 500 kb or less, and the primers should be extended for a suitable period of time at this temperature.) ⑤ Final extension temperature: 72 ℃ for about 10 min. Steps ② to ④ above are PCR cycling steps, and a typical PCR reaction can be set up for 30 to 40 cycles. D Finish the reaction by storing the PCR product at 4 笆 for electrophoretic detection or at -20 °C for long-term storage. E. Microagarose gel electrophoresis of amplified products. 5 to 10 μL of the product is taken directly for electrophoresis. F Gel staining. G Gel imaging system for scanning gel images. Caveat 1 PTE primers should preferably be stored in anhydrous conditions, which will increase the stability of the primers.2 It is important to use autoclaved ultrapure water for primer dissolution and PCR sample preparation, as some polar electrolytes may affect the rate of dissociation of the OXP protecting groups.3 The actual activation time of a single OXP-modified primer is slightly longer than its theoretical half dissociation time, which will facilitate the effective dissociation of the protective group, and the amplification efficiency of MAF-modified primers in the PCR reaction is lower than that of OXP-modified primers. For more product details, please visit Aladdin Scientific website.
Nucleic acid gel electrophoresis device (BioRad), gel imaging system (Amersham Biosciences), UV spectrophotometer.
Reagents:
① DNA template.
② Gene-specific primers: Primers are different depending on the DNA to be amplified. Common primers and corresponding heat-activated primers can be synthesized in the company, mainly using the traditional solid phase synthesis technology, with the help of modified phosphoramidite reagents, OXP or MAFPTE modifying groups are introduced into the primers (at present, TriLink provides a Clean Amp™ dNTP, which can be used for hot-start PCR).
(iii) DNA polymerase and corresponding PCR buffer (typically 10X PCR buffer), purchased from TaKaRa or Invitrogen as needed (typically the buffer provided contains Mg2
+
(the buffer provided by the company contains Mg2+ , so there is no need to add extra buffer in the experiment).
④ 2.5 mmoI/L dNTP mixture: containing 2.5 mmol/L each of dATP, dCTP, dGTP and dTTP, purchased from TaKaRa or Promega.
⑤ TAE or TBE electrophoresis buffer.
⑤ TAE or TBE electrophoresis buffer. ⑥ Sampling buffer.
⑦ Gel staining solution.