A method for optimizing polymerase chain reaction using graphene quantum dots
A graphene quantum dot and chain reaction technology, applied in the biological field, can solve the problems of poor biocompatibility and large amount of graphene oxide used, and achieve the effects of improved yield and specificity, convenient storage, and simple preparation steps
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Embodiment 1
[0019] Example 1. Method for optimizing polymerase chain reaction using graphene quantum dots
[0020] Step 1: Preparation of graphene quantum dot aqueous solution
[0021] The graphene quantum dot aqueous solution is based on the graphene oxide aqueous solution synthesized by the Hummers method as the starting material, and the Photo-Fenton reaction is used, that is, the H 2 O 2 As oxidant, Fe 3+ It is a catalyst prepared under ultraviolet light irradiation. The product is dialyzed in ultrapure water for 2 days to remove unreacted H 2 O 2 With the small molecules produced by the reaction, a pure graphene quantum dot aqueous solution is obtained. The specific preparation method can refer to the literature (Photo-Fenton Reaction of Graphene Oxide: A New Strategy to Prepare Graphene Quantum Dots for DNA Cleavage. ACSNano, 2012, 6(8), 6592-6599).
[0022] Step 2: Mix the graphene quantum dot solution with the PCR amplification reaction system according to the optimized ratio
[0023] The...
Embodiment 2
[0028] Example 2. Graphene quantum dots improve PCR reaction sensitivity and yield
[0029] Experimental reagents and materials:
[0030] Primer 1: 5'-CGCTAACGGATTCACCAC-3' (SEQIDNO.1);
[0031] Primer 2: 5'-CACGGAAACCGAAGACCA-3' (SEQ ID NO. 2).
[0032] Graphene quantum dot aqueous solution (0.1ng / μl and 1ng / μl), ultrasound 1min at room temperature before use. See the following steps for other reagents.
[0033] PCR conditions: (1) preheating at 94°C for 5 min, (2) denaturation at 95°C for 30s, (3) annealing at 52°C for 30s, (4) extension at 72°C for 30s, steps (2) to (4) repeat 36 cycles, ( 5) Extend at 72°C for 5 min, (6) Cool down at 12°C for 10 min.
[0034] Steps:
[0035] The first step: Screen the minimum amount of DNA template for PCR. Different amounts of DNA templates were added to the PCR system for reaction. The template quantity is: 1.75×10 -2 , 1×10 -2 , 2.5×10 -3 , 1.75×10 -3 , 1×10 -3 , 2.5×10 -4 , 1.75×10 -4 , 1×10 -4 , 2.5×10 -5 , 1.75×10 -5 , 1×10 -5 , 2.5×10 -6 ng...
Embodiment 3
[0039] Example 3. Graphene quantum dots improve PCR product specificity
[0040] Step 1: Add 0.25 ng of DNA template to the PCR system for reaction, replace the DNA polymerase with ExTaq enzyme, the other components of the PCR system are the same as step one in Example 2, make up to 25 μl of sterile water.
[0041] Step 2: Take the amplified product in Step 1 as a template, dilute it by 10 times, add 1 μl to the PCR system, and add different amounts of GQDs for the second round of PCR reaction. The amounts of GQDs are: 0, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5 ng, the other components are the same as in step 1, and sterile water is made up to 25 μl.
[0042] Perform agarose gel electrophoresis on the amplified DNA products, and the experimental results are as attached figure 2 Shown. It can be seen from the figure that as the amount of GQDs added increases, non-specific bands in the product gradually decrease. The optimization effect is best when the final concentration of graphene quantum d...
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