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背景:血清淀粉样蛋白 A 转录激活因子基因启动子区鸟嘌呤和胞嘧啶的含量偏高,常规聚合酶链式反应扩增不易成功.目的:探索提高血清淀粉样蛋白 A 转录激活因子基因启动子区富含鸟嘌呤和胞嘧啶的 DNA 模板聚合酶链式反应扩增方案及优化的扩增体系.方法:提取 THP-1细胞基因组 DNA 作为模板,通过97℃高温预变性及最佳退火温度的探索优化聚合酶链式反应的反应条件,在聚合酶链式反应扩增体系中添加不同浓度的增强剂二甲基亚砜改善反应体系.建立提高血清淀粉样蛋白 A 转录激活因子基因富含鸟嘌呤和胞嘧啶的启动子区聚合酶链式反应扩增效率的方法,同时评估不同浓度二甲基亚砜对聚合酶链式反应扩增结果的影响.结果与结论:97℃高温预变性使模板 DNA 充分解链,同时提高退火温度至60℃,有利于提高聚合酶链式反应扩增产率;反应体系中添加2%二甲基亚砜可以提高血清淀粉样蛋白 A 转录激活因子基因启动子区聚合酶链式反应产物的特异性和产率,但是鸟嘌呤和胞嘧啶含量不同对增强剂的依赖不一样.结果证实,高温预变性和较高的退火温度可以保证充分解链的模板 DNA 与引物特异性结合,但是 DNA 模板鸟嘌呤和胞嘧啶含量的不同对增强剂二甲基亚砜的依赖有差异.
배경:혈청정분양단백 A 전록격활인자기인계동자구조표령화포밀정적함량편고,상규취합매련식반응확증불역성공.목적:탐색제고혈청정분양단백 A 전록격활인자기인계동자구부함조표령화포밀정적 DNA 모판취합매련식반응확증방안급우화적확증체계.방법:제취 THP-1세포기인조 DNA 작위모판,통과97℃고온예변성급최가퇴화온도적탐색우화취합매련식반응적반응조건,재취합매련식반응확증체계중첨가불동농도적증강제이갑기아풍개선반응체계.건립제고혈청정분양단백 A 전록격활인자기인부함조표령화포밀정적계동자구취합매련식반응확증효솔적방법,동시평고불동농도이갑기아풍대취합매련식반응확증결과적영향.결과여결론:97℃고온예변성사모판 DNA 충분해련,동시제고퇴화온도지60℃,유리우제고취합매련식반응확증산솔;반응체계중첨가2%이갑기아풍가이제고혈청정분양단백 A 전록격활인자기인계동자구취합매련식반응산물적특이성화산솔,단시조표령화포밀정함량불동대증강제적의뢰불일양.결과증실,고온예변성화교고적퇴화온도가이보증충분해련적모판 DNA 여인물특이성결합,단시 DNA 모판조표령화포밀정함량적불동대증강제이갑기아풍적의뢰유차이.
BACKGROUND: Higher levels of guanine and cytosine in the promoter region of serum amyloid A-activating transcription factor are apt to cause unsuccessful PCR reactions. OBJECTIVE: To optimize the conditions and amplification system for PCR amplification in the promoter region of serum amyloid A-activating transcription factor with rich guanine and cytosine. METHODS: THP-1 cel genomic DNA was extracted as a template, to optimize amplification conditions through 97 ℃denaturation and the best annealing temperature. In addition, different concentrations of dimethyl sulfoxide as an enhancer were added to improve the PCR amplification system. The method of promoting PCR efficiency in the promoter region of serum amyloid A-activating transcription factor with rich guanine and cytosine was established, and meanwhile, the effects of different-concentration dimethyl sulfoxide on PCR results were assessed. RESULTS AND CONCLUSION: During the PCR cycling, the denaturing temperature of 97 unlocked the double-stranded and the annealing temperature increased to 60 specificity and efficiency. The use of 2% dimethyl sulfoxide was shown to improve the yield and specificity of PCR products in the promoter region of serum amyloid A-activating transcription factor. However, templates with different guanine and cytosine content had different dependence on the enhancer. It has been aproved that, during the PCR cycling, high denaturing temperature and high annealing temperature at which the primers bind to their specific template DNA unlocking the double-stranded are critical for specificity and efficiency of the PCR amplification. Templates with different guanine and cytosine contents, however, depend on dimethyl sulfoxide in different manners.