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Sheng Wu Gong Cheng Xue Bao. 2018 May 25;34(5):785-793. doi: 10.13345/j.cjb.170405.

[Selection of DNA aptamers to cervical intraepithelial neoplasia by SELEX].

[Article in Chinese; Abstract available in Chinese from the publisher]

Author information

1
Biochemistry and Molecular Biology Laboratory, Lanzhou University, Lanzhou 730000, Gansu, China.
#
Contributed equally

Abstract

in English, Chinese

An in vitro synthesized random ssDNA library was subjected to 12 rounds of selection against anti-screening cells and sieving cells by SELEX. Normal and inflammatory cervical exfoliation cells were selected as anti-screening cells, and the cervical exfoliation cells of low-grade squamous intraepithelial lesion (CIN1), high-grade squamous intraepithelial lesion (CIN2, CIN3) and cervical carcinoma were selected as sieving cells during the screening process. Then, the highly specific aptamer CIN-Ap4 was established by the analysis of the specificity, affinity and cell immunofluorescence, which can be used as biomarker for Cervical Intraepithelial Neoplasia. Prime Premier 5.0 was applied to design a random ssDNA library. According to the fixed sequence at both ends of the library, a pair of primers were designed and synthesized. At the same time, the optimal annealing temperature, cycle times and primer concentration ratio of PCR procedure were selected. The results under the optimal condition are shown as follows. In the 50 μL reaction system, the optimum reaction conditions of symmetry PCR are as follows: annealing temperature is 49.5 ℃, number of cycles is 15. The optimal reaction conditions of indirect asymmetric PCR are as follows: the primer concentration ratio is 80:1, and the number of cycles is 35. The experiment proves that the oligonucleotide library is constructed successfully, and the highly specific dsDNA and ssDNA can be obtained under optimal PCR conditions with good repeatability, which establishes the foundation for the further exploration and experimentation.

KEYWORDS:

biomarker; cervical intraepithelial neoplasia (CIN); optimize of PCR conditions; systematic evolution of ligands by exponential enrichment (SELEX)

PMID:
29893086
DOI:
10.13345/j.cjb.170405
[Indexed for MEDLINE]
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