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An amplified product, one-to-one technology, applied in the field of molecular biology, can solve the problems of limited amplification efficiency and failure to break through the basic mode, and achieve the effects of prolonging the linear accumulation period, high rigor, and improving rigor
Active Publication Date: 2020-11-06
SUN YAT SEN UNIV
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[0003] Since the invention of PCR technology, although various improved and derived methods have emerged in an endless stream, they have failed to break through its basic model, that is, the amplification of each target gene is carried out through a pair of upstream and downstream primers complementary to the template.
Conventional PCR uses a pair of primers for amplification, and each cycle can only produce twice the amplification product at most, so its amplification efficiency is limited
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Embodiment 1
[0027] Multiplex PCR amplification template DNA:
[0028] Taking the artificially synthesized template of 69 bp as the amplification target, the following primers were designed according to the principle of multiple primer and universal primer design in the technical scheme (Table 1).
[0029] The reaction conditions are as follows:
[0030] The total volume of the PCR reaction system is 25 μL, including 10 U of SD DNA polymerase, 1× SD reaction buffer, and 3.5 mM MgCl 2 , 0.4mM dNTPs, 30nM upstream and downstream multiplex primers Fm and Rm, 1μM upstream and downstream universal primers FUP and RUP, 0.5μL SYBR Geen I (20x), 2μL template. Sterile water was used as negative control.
[0031] The PCR amplification program was: denaturation at 92°C for 2 min, followed by denaturation at 92°C for 20 s, annealing at 56°C for 30 s, extension at 68°C for 20 s, and 25 cycles. Take 5 μL of the above amplification solution and add 1 μL of 6x loading buffer, mix well and take 5 μL for...
Embodiment 2
[0039] Real-time quantitative multiplex PCR detection of Salmonella:
[0040] Multiple primers and universal primers were designed with the ttr gene of Salmonella as the detection target (Table 2).
[0041] The reaction conditions are as follows:
[0042] The total volume of the PCR reaction system is 25 μL, including 10 U of SD DNA polymerase, 1× SD reaction buffer, and 4 mM MgCl 2, 0.4mM dNTPs, 30nM upstream and downstream 2-fold primers F2 and R2 each, 1μM upstream and downstream universal primers FUP and RUP, 0.5μL SYBR Geen I (20x), 2μL template. Sterile water was used as negative control.
[0043] The PCR amplification program was: denaturation at 92°C for 2 min, followed by denaturation at 92°C for 30 s, annealing at 55°C for 30 s, extension at 68°C for 1 min, and fluorescence detection at the extension section, 35 cycles.
[0044] Table 2 List of primer sequences for detection of Salmonella ttr gene
[0045]
[0046] Such as Figure 4 as shown, Figure 4 It is...
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Abstract
The invention provides a multi-PCR (Polymerase Chain Reaction) amplification method, that is, a multi-primer (low concentration) consisting of a pair of 5'-ends and a plurality of identical sequences with same universal primers and a pair of universal primers (high concentration) are amplified in a same PCR, and DNA (Deoxyribonucleic Acid) polymerase used in the amplification is thermal-resistant polymerase with a chain substitution function. The multi-primer sequence comprises two parts, namely a 3'-end and a target gene which are supplemented by each other, and the 5'-end carries at least one sequence identical to the universal primers; the numbers of universal primers carried by the 5'-ends of upstream multi-primers and downstream multi-primers are identical or different, and the sequences of upstream universal primers and downstream universal primers are also identical or different. By adopting the method, a plurality of amplification products are generated through one time of circulation, and in addition to target amplification sequences, each amplification product comprises at least one upstream universal primer sequence and one downstream universal primer sequence. As more than two times of amplification products are generated through one time of circulation, the amplification efficiency and detection sensitivity of PCR are remarkably improved, and the method is applicable to detection on low-template number genes by means of common molecular biology labs and medicinal nucleic acid assays.
Description
technical field [0001] The invention relates to the technical field of molecular biology, in particular to a multiplex PCR amplification method. Background technique [0002] As a method for rapidly amplifying specific genes in vitro, PCR technology has been widely used in various fields due to its high specificity and sensitivity. The key to the PCR method is to design a pair of oligonucleotide primers with high stringency according to the target gene, and realize rapid amplification through the enzymatic reaction of DNA polymerase. [0003] Since the invention of PCR technology, although various improved and derived methods have emerged in an endless stream, they have failed to break through the basic model, that is, the amplification of each target gene is carried out through a pair of upstream and downstream primers complementary to the template. Conventional PCR uses a pair of primers for amplification, and each cycle can only produce twice the amplification product at...
Claims
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