Team:USTC/Proof Of Concept

USTC-iGEM-Proof_Of_Concept


Hello, Welcome to PROOF OF CONCEPT PAGE

Exploration of the conditions of asymmetric PCR


    The template we design consists of NA and AP single chains and aptamer with block. The NA and AP strands we obtained from the company existed are in the form of double chains, but we only need to obtain the single chain of NA and AP through asymmetric PCR. Similar to symmetric PCR, we add templates, primer 1, primer 2, dNTPs, ultra-pure water and DNA polymerase. The template chains will unwind at high temperature. Then the primers can bind to the DNA double strands at annealing temperature, and the primers can extend with the help of DNA polymerase at elongation temperature. However, in asymmetric PCR, the amount of the two primers we add is different from symmetric PCR. According to previous research, more primer 1 should be added. In this way, when primer 2 all extends to dsDNA, the remaining primer 1 will find the binding site and extend a large number of ssDNA required.
    However, the selection of the proportion and amount of two primers is also a problem. If we add too many primer 1 and only few primer 2, there will be few complementary chains to produce the target single strands, resulting in a slow reaction rate. If we add very few prime 1, the number of ssDNA we obtain will decrease. So, it is a problem how we choose proper ratio and number of primers 1 and 2.In order to explore the most perfect proportion and number of primers, we first tried asymmetric PCR with primer ratios of 10:1, 15:1, 20:1, 25:1 and 30:1 at template concentration, and then detected the yield of ssDNA by using the brightness of gel electrophoresis. It was found that the yield of ssDNA was the highest at 20:1 (Fig 1). In addition, the ratio of 20:1 primer was 4:1/5, 5:1/4, 8:2/5, 10:1/2 for the experiment, and it was found that the yield of single strand was the largest in the ratio of 20:1 (Fig 2). In order to determine the best annealing temperature, we set 54˚C, 56 ˚C, 58 ˚C, 60 ˚C and 62 ˚C (Fig 3). The gray scale measured by ImageJ showed that at 58 ˚C we could get best result (Fig 4). Through those confirmatory experiments above, we confirmed the final condition of asymmetric PCR (Fig 5).


Fig 1. NA ssDNA 10:1 15:1 20:1 25:1 30:1（6-10 bands）

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Fig 2. NA ssDNA 4:1/5，5:1/4，8:2/5，10:1/2，20:1 (2-6 bands)

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Fig 3. NA ssDNA 54˚C 56 ˚C 58 ˚C 60 ˚C 62 ˚C (2-6 bands)

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Fig 4. The gray scale measured by ImageJ

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Fig 5. The most suitable conditions for asymmetric PCR reaction we have explored

Results of asymmetric PCR


    In order to obtain the NA and AP single chains we need, we conducted asymmetric PCR according to the conditions explored before. However, because the movement of single chain can not be separated by marker alone, we validated the products of asymmetric PCR. We put the NA ssDNA and its complementary strands together, 98 ℃ for 5min, and cooled them naturally in a water bath to get recombinant double chains. So was the AP chain. We performed an electrophoresis to compare the original NA and AP double strands and the recombinant double strands. (Fig 6)


Fig 6 original double strands and recombinant double strands

    The left two channels are original AP and NA chains, the next two channels are recombinant chains. From the figure we can find that the two NA chains are at the same position, the two AP chains as well. It proves that the single strands obtained by asymmetric PCR are correct.

Exploration of qPCR


    In order to transform the detection results of our detection means from chemical signal to fluorescence signal, we started the real-time fluorescence quantitative PCR (qPCR) technology experiment. We added Buffer A, Buffer B, Recombinase polymerase, our template and the prime to the reaction system.Click here to see the protocol of qPCR.

1. The order of the adding samples
    We tried a variety of sampling sequences to determine in which way the data is more stable.

    a. BufferA+Enzyme+ddH20+Template+Primer1+Primer2+Probe+BufferB
    b. First add BufferA+Enzyme+ddH20+Primer1+Primer2+Probe.
        Then Template+BufferB
    c. First add BufferA+Enzyme+ddH20+Primer1+Primer2
        Then Template+Probe.+BufferB (mix)
    d. First add BufferA+Enzyme
        Then ddH20+Template+Primer1+Primer2+Probe+BufferB （mix）
    e. First add BufferA+Enzyme+ddH20+Template
        Then Primer1+Primer2+Probe+BufferB （mix）
    f. First add BufferA+Enzyme+ddH20+Template+Probe
        Then Primer1+Primer2 +BufferB （mix）

    We found that order c is the best way. It can basically ensure that all samples react together and will not produce fluorescence signals without mixing. When measuring the protein’s concentration, the protein is also mixed with bufferB.

2. The concentration of the template
    In order to solve the problem of template concentration in our qPCR experiment, we conducted a series of experiments to explore the appropriate concentration of template. We conducted a comparative experiment with different concentrations of aptamers and without aptamers. (Fig 7)


Fig7 different concentrations of template chain A:10-8mol/L B:10-9mol/L C:10-10mol/L D:10-11mol/L E:10-12mol/L F:10-13mol/L

    The concentrations of the template chains we chose are still from 10-8 to 10-13 mol/L. In the groups with aptamers, we added twice the template concentration of aptamers to ensure more template bind aptamers. The gray curve is aptamer-free while the red curve is with aptamer, and the blue curve is blank group. We can easily make a distinction between the curves representing aptamer and aptamer-free. At any concentration, the curves representing aptamer-free is above the curves representing aptamer, while the blank control group is the lowest in the figures. Both curves are above the blank control group, proving that our data are valid. It means that the aptamer can successfully bind to the template strand, and the block can inhibit the DNA polymerase from completing the template strand. The amplification reaction is blocked and reflected in the fluorescence signal intensity.
    Those figures above preliminarily prove the feasibility of our experiment. According to experiment data, when the concentration of template is 10-10mol/L, the comparison between the presence and absence of aptamers is obvious enough, and there is no need to select template chain with a higher concentration. Therefore, further experiments on tau protein were carried out under the condition of template chain of 10-10 mol/L. In addition, it is proved that the experiment can achieve good results under the condition of two-equivalent aptamer, so the two-equivalent aptamer was also selected in the subsequent experiments.

Detection


1. ddH2O
    In order to prove our design, we carried out qPCR reaction of different concentrations of tau protein in ddH2O system. The experimental results shows that the fluorescence intensity will increase as the Tau protein concentration grows(Fig 8).


Fig 8 different concentrations of tau protein in an aqueous solution

2. Serum
    To verify that our design can be used in a real blood test, we replaced water with a serum buffer. Under the same condition, we got the figure of different concentrations of tau protein and fluorescence intensity. (Fig 9)


Fig 9 different concentrations of tau protein in a serum buffer

Software and hardware


     In order to achieve more convenient detection, we designed supporting hardware and software. The hardware is used to detect and output the fluorescence signal, and the software is used to receive the signal for processing. We tested the software and optimized the interface, which greatly improved the application ability of our scheme.     (portal: Software)     (portal: Hardware)

Summary

     In short, we have adopted many means to prove that our design is feasible. We hope this detection method can be used to practice and contribute to the conquer of diseases.