Multiplex polymerase chain reaction method and applications thereof
A technology of chain reaction and polymerase, applied in the field of multiplex polymerase chain reaction, can solve the problems of unsuitable optimization scheme, inability to meet the practical application of multiplex PCR, high time cost and economic cost
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Embodiment 1
[0153] Example 1 The effect of adding AuNPs at different primer concentrations on the multiplex PCR amplification efficiency of human genome template and λ DNA
[0154] The composition of the PCR solution system is shown in Table 3:
[0155] table 3
[0156]
[0157] PCR steps and conditions: (1) Pre-denaturation at 95°C for 5 minutes; (2) Pre-denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, extension at 72°C for 1 minute and 30 seconds; repeat 40 cycles; (3) Extension at 68°C 10 minutes.
[0158] Electrophoresis analysis conditions: agarose gel electrophoresis (3%), voltage: 80V, electrophoresis time 3-4h.
[0159] result
[0160] In this example, 8 pairs of primers were used to simultaneously perform multiple amplification of 8 different gene fragments on the human genome template and λ DNA, and the reaction conditions are shown in Table 3. Different concentrations of primer mixes were added to multiplex PCR reactions, each containing negative withou...
Embodiment 2
[0163] Example 2 Effects of Different AuNPs Concentrations on Human Genome Template and Lambda DNA Multiplex PCR Amplification Efficiency
[0164] The composition of the PCR solution system is shown in Table 4:
[0165] Table 4
[0166]
[0167] The primer concentration of each group of primers was set to 200nM (single primer final concentration). Different amounts of AuNPs were added to the multiplex PCR reaction system, respectively 0, 1, 3, 5, 7, 9, 10, 11, 12, 13, 14, 15, 16 and 17 μL. The total reaction volume was 25 μL.
[0168] result
[0169] image 3 is the result of electrophoretic analysis of the reaction mixture obtained from the human genome template, from image 3 It can be seen that in the case of not adding AuNPs, only 4 bands of the target product are visible in the multiplex PCR products, and multiple bands of non-specific products can be seen at the same time, indicating that the multiplex PCR is specific without adding AuNPs. The sensitivity is low...
Embodiment 3
[0171] Example 3 Effect of AuNPs on Human Genome Template and Lambda DNA Multiplex PCR Amplification Sensitivity under Different Template Concentrations
[0172] Using human genomic DNA and lambda DNA as templates, 8 pairs of primers were used to simultaneously perform multiple amplification of 8 different gene fragments on the template. The amounts of DNA templates were 175ng, 25ng, 5ng and 1ng respectively. The remaining reaction components are the same as in Table 4, and the PCR reaction conditions are shown in Example 1.
[0173] result
[0174] Figure 5 is the result of electrophoresis analysis of the reaction mixture obtained under different concentrations of human genome template, from Figure 5 It can be seen that when no AuNPs are added, the number of specific product bands successfully amplified by multiplex PCR decreases significantly with the decrease of the template amount, and all target bands cannot be successfully amplified even when the template amount is ...
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