Method for detecting DNA tetrahedron through utilizing capillary electrophoresis
A capillary electrophoresis and tetrahedron technology, which is applied in the field of biological analysis, can solve the problems of inability to detect DNA tetrahedrons in trace amounts and conveniently, and achieve the effects of simple operation, high repeatability and extended application.
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Embodiment 1
[0033] 1. Synthesis of DNA tetrahedron:
[0034] Centrifuge the purchased (Sangon (Shanghai, China)) DNA single strands (S1, S2, S3, S4) with a centrifuge (8000r / min, 3min), then add TM buffer to make the DNA single strand concentration 100μM . Mix the four single strands forming the DNA tetrahedron at equimolar concentrations in TM buffer to form a sample with a final DNA concentration of 2 μM; heat the prepared sample in a water bath at 95°C for 10 minutes, then rapidly cool down to 4 ℃, and kept in a refrigerator at 4 ℃ for 3 hours, the DNA tetrahedral structure can be obtained.
[0035] 2. Synthesis of linear polyacrylamide:
[0036] Weigh 5g of acrylamide to make a 50mL solution, and ultrasonicate for 15min; add 16μL of tetramethylethylenediamine to the 50mL of acrylamide solution, quickly add 180μL of 10% ammonium persulfate solution, stir evenly, and seal it after ultrasonic for 15min. Polymerization was carried out at (4°C, 25°C, 50°C) for 24 hours.
[0037] 3. Tak...
Embodiment 2
[0041] According to the method of Example 1, the optimum sieving agent concentration and preparation conditions were selected, and other electrophoresis conditions were explored respectively, and DNA tetrahedrons and various types of DNA strands, including: single strand 4, Double strand (strand 1+strand 4), double strand (strand 3+strand 4) were detected. First, DNA tetrahedron and double strand (strand 1+strand 4) are taken as examples for investigation. From image 3 It can be seen that the migration time of DNA decreases with the increase of the buffer pH value, presumably because the higher pH value will promote the increase of the negative charge on the inner wall of the capillary, and the electroosmotic flow will increase. Better detection can be achieved when the pH value is 9.3. Under the condition of optimal buffer pH value, different buffer concentrations of 15, 20, 25, 30 and 35mmol L were selected -1 , when the concentration is 25mmol L -1 A maximum detection ...
Embodiment 3
[0043] According to the method of Example 1, a DNA tetrahedron was synthesized by using single-labeled strand 4, and the complementary strand of strand 4 was added during the synthesis process to interfere with the synthesis of the tetrahedron, resulting in a double strand formed by complementary pairing of strand 4 and strand 6. Capillary electrophoresis is used for detection, and the peaks of DNA tetrahedron and DNA double strand can be determined respectively. Such as Figure 7 , with the increase of the concentration of chain 6, the peak area of DNA tetrahedron decreases, which proves the successful synthesis of DNA tetrahedron. Since a new peak was generated after the addition of complementary strand 6, it was speculated that strand 6 could be used to destroy the DNA tetrahedral structure. The inference of Example 1 was further verified.
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