农业科学与技术:英文版
農業科學與技術:英文版
농업과학여기술:영문판
Agricultural Science & Technology
2012年
2期
273-275,329
,共4页
曹立亭%许李丽%万向%王秋菊%马跃
曹立亭%許李麗%萬嚮%王鞦菊%馬躍
조립정%허리려%만향%왕추국%마약
随机单链DNA文库%正交试验设计%PCR%体系优化
隨機單鏈DNA文庫%正交試驗設計%PCR%體繫優化
수궤단련DNA문고%정교시험설계%PCR%체계우화
Random single-stranded DNA pool%Orthogonal experimental design%Polymerase chain reaction%System optimization
[目的]对SELEX技术中ssDNA文库的PCR扩增条件进行优化。[方法]采用L16(45)正交试验设计在4个水平上对影响单链随机DNA文库PCR反应体系的Mg2+浓度、dNTP浓度、TaqDNA聚合酶含量、引物浓度和随机单链DNA文库量5个重要因素进行了优化,同时对PCR反应的退火温度和循环次数进行了优化,确立最优反应体系和扩增程序。[结果]20μlPCR反应体系及反应程序中各因素优化组合为:10×Buffer2.0μl,随机ssDNA文库0.5ng,Mg2+2.5mmol/L,dNTP Mixture0.25mmol/L,上下游引物各0.6μmol/L,TaqDNA聚合酶1.5U;退火温度为68℃,最佳循环数为12。此反应系统下,随机ssDNA文库PCR扩增谱带清晰、稳定、特异性高。[结论]为SELEX技术中筛选到特异性更强的适配子奠定了基础。
[目的]對SELEX技術中ssDNA文庫的PCR擴增條件進行優化。[方法]採用L16(45)正交試驗設計在4箇水平上對影響單鏈隨機DNA文庫PCR反應體繫的Mg2+濃度、dNTP濃度、TaqDNA聚閤酶含量、引物濃度和隨機單鏈DNA文庫量5箇重要因素進行瞭優化,同時對PCR反應的退火溫度和循環次數進行瞭優化,確立最優反應體繫和擴增程序。[結果]20μlPCR反應體繫及反應程序中各因素優化組閤為:10×Buffer2.0μl,隨機ssDNA文庫0.5ng,Mg2+2.5mmol/L,dNTP Mixture0.25mmol/L,上下遊引物各0.6μmol/L,TaqDNA聚閤酶1.5U;退火溫度為68℃,最佳循環數為12。此反應繫統下,隨機ssDNA文庫PCR擴增譜帶清晰、穩定、特異性高。[結論]為SELEX技術中篩選到特異性更彊的適配子奠定瞭基礎。
[목적]대SELEX기술중ssDNA문고적PCR확증조건진행우화。[방법]채용L16(45)정교시험설계재4개수평상대영향단련수궤DNA문고PCR반응체계적Mg2+농도、dNTP농도、TaqDNA취합매함량、인물농도화수궤단련DNA문고량5개중요인소진행료우화,동시대PCR반응적퇴화온도화순배차수진행료우화,학립최우반응체계화확증정서。[결과]20μlPCR반응체계급반응정서중각인소우화조합위:10×Buffer2.0μl,수궤ssDNA문고0.5ng,Mg2+2.5mmol/L,dNTP Mixture0.25mmol/L,상하유인물각0.6μmol/L,TaqDNA취합매1.5U;퇴화온도위68℃,최가순배수위12。차반응계통하,수궤ssDNA문고PCR확증보대청석、은정、특이성고。[결론]위SELEX기술중사선도특이성경강적괄배자전정료기출。
[Objective] This study aimed to optimize the PCR amplification conditions for random ssDNA pool in SELEX technology. [Method] L16(45) orthogonal experimental design was adopted for optimization of five important factors affecting PCR reaction system for random single-stranded DNA pool including Mg2+ concentration, dNTP concentration, amount of Taq DNA polymerase, primer concentration and amount of random single-stranded DNA pool at four levels. Meanwhile, the annealing temperature and number of PCR reaction cycles were optimized to establish the optimal reaction system and PCR procedure. [Result] The optimal combination of PCR reaction system for random ssDNA pool was obtained, with a total system volume of 20 μl containing 2.0 μl of 10 × Buffer, 0.5 ng of random ssDNA pool, 2.5 mmol/L Mg2+, 0.25 mmol/L dNTP Mixture, 0.6 μmol/L upstream and downstream primers and 1.5 U of Taq DNA polymerase; the optimal annealing temperature was 68 ℃ and the optimal number of cycles was 12. Under the above conditions, clear and stable bands with high specificity for random ssDNA pool were amplified. [Conclusion] This study laid the foundation for selection of parameters with higher specificity in SELEX technology.