Nucleic acid aptamer screening method
A screening method and nucleic acid aptamer technology, which can be used in biochemical equipment and methods, and the determination/inspection of microorganisms, and can solve problems such as no candidate nucleic acid aptamer screening methods.
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Embodiment 1
[0032] To the nucleic acid sample to promote nucleic acid samples to form a secondary structure, amplification and sequencing of nucleic acid samples, including the following steps:
[0033] (1) The genomic DNA was extracted from phenol-chloroform from the cultured human K562 cell line and dissolved in TE buffer. The nucleic acid sample to be tested is divided into two groups, each containing 10 μl of a total of 100 ng of genome nucleic acid.
[0034] (2) Add a final concentration of 1 μm MnCl to the above-mentioned nucleic acid sample 2 The solution enables both to mix; another group of isometric water.
[0035] (3) The MDA total genome is added to the MDA total genomic, 4 mmDNTP, 1U / μl29 polymerase, and corresponding buffer, respectively, the reaction conditions for the MDA total genome, and the reaction conditions were carried out on a 50 μm random primer, 4 mmdNTP, 1 U / μL29 polymerase and corresponding buffer.
[0036] (4) After the amplification, the two sets of samples wer...
Embodiment 2
[0040] According to the method of Example 1, a sequence of artificially synthetic nucleic acids were screened by a sequence of nucleic acids.
[0041]
[0042] 2 μl of concentration of 100 μm Pb (NO) was added to a nucleic acid sample of 198 μL of concentration of 20 μm (NO 3 ) 2 The solution allows it to fully react. Another group of sample nucleic acid concentrations are maintained, but add the same molar NANO 3 Solution.
[0043] The amplification of the primary ionic nucleic acid sample set and the non-lead ion-free nucleic acid sample group was amplified, and the PCR (polymerase chain reaction) was amplified. The PCR reaction system contains 400 μm DNTPs, 2 μm primers, 0.025 unit / μl RTAQ and 1 μL of the template in 50 μl of 1 × PCR buffer. The process of PCR is denaturation (92 ° C, 15S), annealing (58 ° C, 30S), extending (72 ° C, 20S), a total of 14 cycles, and finally extends at 72 ° C for 5 min.
[0044] After amplification, the two groups of samples were subjected to...
Embodiment 3
[0047] According to the method of Example 1, a PDS nucleic acid aptamer was performed on a natural nucleic acid sequence from human lymphocytes. The final concentration of 1 μm PDS was added to a nucleic acid sample having a concentration of 20 μm to sufficiently reacted. Another group of sample nucleic acid concentrations is maintained, but is added to the same amount of water.
[0048] The amplification of the PDS-containing nucleic acid sample group and the Nucleic acid sample group containing the PDS were amplified by PCR (polymerase chain reaction). The PCR reaction system contains 400 μm DNTPs, 2 μm primers, 0.025 unit / μl RTAQ and 1 μL of the template in 50 μl of 1 × PCR buffer. The process of PCR is denaturation (98 ° C, 15S), annealing (58 ° C, 30S), extending (72 ° C, 90S), a total of 14 cycles, and finally extends at 72 ° C for 5 min.
[0049] After amplification, the two groups of samples were subjected to ultrasound interruption and ligation enzyme connection sequenc...
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