Detection probe, kit and direct detection method of telomerase activity
A technology for detecting probes and telomerase, which is used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve real-time detection, unique optoelectronic characteristics, and easy real-time detection.
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Embodiment 1
[0044] Embodiment 1: the preparation of detection probe
[0045] Step (1): Take 200uL 50nM gold nanoparticle solution with a diameter of 5nm, add 50uL 5×TBE solution and 2uL 100uM sulfhydryl-modified telomerase primer sequence (SH-ttttttaatccgtcgagcagagtt), use an oscillator to shake evenly, put React on a small shaker at 37°C and 500rpm for 10h.
[0046] Step (2): Add salt to the product obtained in step (1) for aging, use 3M NaCl solution, add the sample in 5 times, add 10uL each time, the final concentration of NaCl in the sample is 500mM, and continue to place it on a small shaker at 37°C , 500rpm reaction 10h.
[0047] Step (3): Use a balance to balance the sample obtained in step (2), and use a refrigerated centrifuge to centrifuge at 15,000 rpm for 30 minutes at 25°C, remove the supernatant and retain the precipitate, redissolve with 50-100uL ultrapure water, and repeat three times to obtain the detection Store the probe in a 4°C refrigerator.
[0048] Among them, th...
Embodiment 2
[0050] Example 2: Preparation of DNA-AuNPs complex
[0051] Use telomerase extracted from Hela cells to activate and extend the telomeres enriched on the surface of gold nanoparticles, and the operation steps are as follows:
[0052] Step (1): Use mild CHAPS lysate (aladdin) to lyse the cultured Hela cells and extract the telomerase therein. According to the operating instructions of the kit, in the cell sediment obtained by centrifugation, about 10 6 Add 200uL cell lysate to each cell, oscillate, lyse, suck it out and put it into a 1.5mL centrifuge tube; place the centrifuge tube in an ice bath, place it in a 4°C refrigerator for 10min, use a refrigerated centrifuge at 4°C, centrifuge at 15000rpm for 30min, and take out the supernatant. Pack and store in -80°C refrigerator.
[0053] Step (2): Take 50uL of the detection probe solution (about 20nM) prepared in Example 1, add 10uL TRAP solution, 2uL100mM dNTP solution and 28uL ultrapure water, mix well, add the telomerase extra...
Embodiment 3
[0056] Example 3: Detection of extension products of DNA-AuNPs complex by nanopore sensor
[0057] According to the method of Example 2, through the regulation of SDS denaturant, prepare the telomerase extension telomeric DNA sequence at different times to produce DNA-golden sphere complexes with different chain lengths, and use the resulting product using a 20nm silicon nitride nanopore The sensor uses a bias voltage of 600mV to detect DNA-AuNps at different time gradients. After the telomeres on the surface of the AuNps are extended by telomerase, the surface of the DNA-AuNps will be induced. The charge carried and the hydrated particle size change. These changes in properties can be detected by the change of the highly sensitive nanopore ion current signal and the generated current blocking signal to distinguish products under different time gradients. Enzyme activity and monitoring of the kinetic process of telomeric sequence extension on the surface of gold spheres.
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