CAJ | 학술논문

目的优化三阴乳腺癌(TNBC)适配子筛选中单链DNA(ssDNA)文库扩增条件,探讨次级ssDNA最佳制备方法.方法构建ssDNA文库,设置不同的模板量(1012、1011、1010、109、108、107、106、105条带)、循环数(9、12、15、18、21、24、27、30)、DNA聚合酶量(0.025、0.050、0.100、0.200、0.400 U/μl)进行聚合酶链反应(PCR)扩增;对比3种制备次级ssDNA的方法,取其中最佳方法进行筛选.结果 20μl体系最优扩增条件为:ssDNA模板量108个条带、扩增循环数为12、DNA聚合酶量为0.100 U/μl;不对称PCR法所得ssDNA量最多.结论确立了ssDNA文库最优扩增条件及次级ssDNA最佳制备方法,为进一步筛选TNBC特异性适配子提供实验基础.
목적 우화삼음유선암(TNBC)괄배자사선중단련DNA(ssDNA)문고확증조건,탐토차급ssDNA최가제비방법.방법 구건ssDNA문고,설치불동적모판량(1012、1011、1010、109、108、107、106、105조대)、순배수(9、12、15、18、21、24、27、30)、DNA취합매량(0.025、0.050、0.100、0.200、0.400 U/μl)진행취합매련반응(PCR)확증;대비3충제비차급ssDNA적방법,취기중최가방법진행사선.결과 20μl체계최우확증조건위:ssDNA모판량108개조대、확증순배수위12、DNA취합매량위0.100 U/μl;불대칭PCR법소득ssDNA량최다.결론 학립료ssDNA문고최우확증조건급차급ssDNA최가제비방법,위진일보사선TNBC특이성괄배자제공실험기출.
Objective To optimize the polymerase chain reaction (PCR) amplification conditions of single-stranded DNA (ssDNA) pool and evaluate different methods for generation of ssDNA in selecting targeted aptamers of triple negative breast cancer (TNBC) by systematic evaluation of ligands by exponential enrichment (SELEX).Methods ssDNA library was synthesized.The initial template concentration (1012,1011,1010,109,108,107,106 and 105 sequences),amplification cycles (9,12,15,18,21,24,27 and 30) and DNA polymerase concentrations (0.025,0.050,0.100,0.200,and 0.400 U/μl) were set up for the amplification of random ssDNA.Efficiency of ssDNA generation was evaluated by comparing three most used methods.Results The optimal ssDNA template concentration was 108 sequences,cycle number was 12 and DNA polymerase concentration was 0.100 U/μl with a total system volume of 20 μl.Asymmetric PCR produced the highest amount of ssDNA.Conclusion The PCR amplification conditions of ssDNA library and the best method to generate ssDNA were established,which laid experimental foundations for the further selection of TNBC targeted aptamers.