Shanghai Haozhuang Instrument Co., Ltd. has developed the Haozhuang (LNB) brand gene amplification instrument, which is energy-saving and environmentally friendly, leading the country. Principle of Gene Amplifier: PCR is the abbreviation of polymerase chain reaction. It refers to the amplification reaction of a specific template (cloned or genomic DNA) catalyzed by an enzyme under the guidance of primers. It simulates the DNA replication process in vivo and is specific in vitro A technique for amplifying DNA fragments has a wide range of applications in molecular biology, including DNA mapping, DNA sequencing, molecular systems genetics, etc. The basic principle of PCR is to use single-stranded DNA as a template and 4 kinds of dNTPs as substrates. In the presence of primers at the 3 'end of the template, the complementary strand is extended with an enzyme. Multiple repeated cycles can obtain a small amount of template DNA Greatly amplified. In a microcentrifuge tube, add two primers complementary to the known sequences at both ends of the DNA fragment to be amplified, an appropriate amount of buffer, a small amount of DNA membrane plate, four dNTP solutions, heat-resistant Taq DNA polymerase, Mg2 + Wait. During the reaction, the above solution is first heated to denature the template DNA at high temperature, and the double-stranded strand is unwound into a single-stranded state; then, the temperature of the solution is reduced, and the synthetic primer is paired with its target sequence at low temperature to form a partial double-stranded strand, called annealing; Then raise the temperature to a suitable temperature. Under the catalysis of Taq DNA polymerase, using dNTP as the raw material, the primer extends in the 5 '→ 3' direction to form a new DNA fragment, which can be used as a template for the next round of reaction. Repeatedly changing the temperature in this way constitutes a cycle consisting of high temperature denaturation, low temperature renaturation and moderate temperature extension, and repeated cycles, so that the target gene can be rapidly amplified. Therefore, the PCR cycle consists of three parts: template denaturation, primer annealing, and thermostable DNA polymerase to catalyze the synthesis of DNA strand extension at an appropriate temperature. 1. Denaturation of template DNA When template DNA is heated to 90 ~ 95 ℃, the hydrogen bond of the double helix structure is broken, and the double strand is unwrapped to become a single strand, called DNA denaturation, so that it can be combined with primers to prepare for the next round of reaction . Denaturation temperature is related to the content of GC in DNA. GC is connected by three hydrogen bonds, while AT is only connected by two hydrogen bonds, so the melting temperature of template with higher GC content is relatively higher. Therefore, the temperature and time required for DNA denaturation in PCR are related to the complexity of the secondary structure of the template DNA and the level of GC content. For the template DNA with high GC content, a certain amount of dimethyl sulfoxide (DMSO) needs to be added in the experiment, and the thermal denaturation temperature in the initial stage of the PCR cycle can be 97 ° C, and the time is appropriately extended, so-called hot start. 2. Annealing of template DNA and primers When the temperature of the reaction mixture is reduced to 37 ~ 65 ℃, oligonucleotide primers hybridize with single-stranded templates to form DNA template-primer complexes. The temperature and time required for annealing depends on the degree of homology between the primer and the target sequence and the base composition of the oligonucleotide. Generally, the concentration of the primer is much higher than the concentration of the template DNA, and because the length of the primer is significantly shorter than the length of the template, during annealing, the pairing speed of the primer and the complementary sequence in the template is faster than that of the template. The speed is much faster, the annealing time is generally 1 ~ 2min. 3. Extension of the primer DNA template-primer complex under the action of Taq DNA polymerase, using dNTP as the reaction raw material, the target sequence as the template, according to the principle of base pairing and semi-retaining replication, synthesize a new complementary to the template DNA strand chain. By repeating the three processes of cycle denaturation-annealing-extension, more "semi-retained replication chains" can be obtained, and this new chain can become a template for the next cycle. The time required for extension depends on the length of the template DNA. At 72 ° C, the synthesis rate catalyzed by Taq DNA polymerase is about 40 to 60 bases / sec. After a round of "denaturation-annealing-extension" cycle, the template copy number doubled. In subsequent cycles, the newly synthesized DNA can serve as a template, so after each cycle, the DNA copy number doubles. Each cycle takes 2 to 4 minutes, and one PCR takes 30 to 40 cycles, about 2 to 3 hours. In the initial stage of amplification, the amount of amplification increased linearly, but when the primer, template, and polymerase reached a certain ratio, the catalytic reaction of the enzyme tended to saturate, and the so-called "platform effect" appeared, that is, the concentration of target DNA product no increase. The three reaction steps of PCR are repeated, and the amount of DNA amplification increases exponentially. The final DNA amplification of the reaction can be calculated by Y = (1 + X) n. Y represents the number of copies of the amplified DNA fragment, X represents the average (Y) amplification efficiency each time, and n represents the number of cycles. The theoretical value of the average amplification efficiency is 100%, but the average efficiency does not reach the theoretical value in the actual reaction. In the early stage of the reaction, the increase in the target DNA fragment is in an exponential form. With the gradual accumulation of PCR products, the amplified DNA fragment no longer increases exponentially, but enters a linear growth period or a stationary phase, that is, a "stasis effect" This effect is called factors such as plateau number, PCR amplification efficiency, the type and activity of DNA polymerase PCR, and competition for non-specific products. In most cases, the arrival of the platform period is inevitable. PCR amplification products can be divided into two parts: long product fragments and short product fragments. The length of the short product fragment is strictly defined between the 5 ends of the two primer chains and is a specific fragment that needs to be amplified. The short product fragment and the long product fragment are formed due to the different template bound by the primer. Taking an original template as an example, in the first reaction cycle, two complementary DNAs are used as the template, and the primer starts from the 3 end For extension, the 5 end is fixed, and the 3 end has no fixed dead point, and the length is different. This is the "long product fragment". After entering the second cycle, in addition to binding to the original template, the primer must also bind to the newly synthesized strand (ie, "long product fragment"). When the primer is combined with the new strand, since the sequence at the 5 end of the template of the new strand is fixed, this is equivalent to a fixed stop at the 3 end of the extended fragment this time, ensuring that the start and stop points of the new fragment are limited to the primer expansion. Within the increasing sequence, a "short product fragment" of consistent length is formed. It is not difficult to see that "short product fragments" are increased by exponential multiples, while "long product fragments" are increased by arithmetic multiples, which is almost negligible, which makes PCR reaction products do not need to be purified, to ensure that enough pure DNA fragments for For analysis and testing.
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