生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2013年
5期
645-649
,共5页
邢玉华%戴素琴%刘体颜%王微%谭俊杰%曲国龙%刘刚%陈惠鹏
邢玉華%戴素琴%劉體顏%王微%譚俊傑%麯國龍%劉剛%陳惠鵬
형옥화%대소금%류체안%왕미%담준걸%곡국룡%류강%진혜붕
达托霉素生物合成基因%高GC含量DNA%PCR添加剂%touch down PCR
達託黴素生物閤成基因%高GC含量DNA%PCR添加劑%touch down PCR
체탁매소생물합성기인%고GC함량DNA%PCR첨가제%touch down PCR
daptomycin biosynthetic gene cluster%GC-rich DNA%PCR additives%touch down PCR
目的::探索高GC含量DNA的PCR扩增条件,为扩增达托霉素生物合成基因簇及拼接奠定基础。方法:在PCR扩增体系中,使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂,并选择合适的PCR循环程序,优化富含GC的DNA的PCR扩增条件。结果:向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量,但会降低其特异性;7-deaza-dGTP可以提高扩增产物的特异性及保真度,但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率,增加扩增的保真度。结论:应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来,并可扩增出长达6 kb的片段,且序列完全正确,可以进行后续拼接。
目的::探索高GC含量DNA的PCR擴增條件,為擴增達託黴素生物閤成基因簇及拼接奠定基礎。方法:在PCR擴增體繫中,使用高保真的聚閤酶及添加不同濃度的DMSO、7-deaza-dGTP等增彊劑,併選擇閤適的PCR循環程序,優化富含GC的DNA的PCR擴增條件。結果:嚮反應體繫中額外添加1%~4%的DMSO可以顯著提高富含GC的DNA的PCR擴增產物量,但會降低其特異性;7-deaza-dGTP可以提高擴增產物的特異性及保真度,但產量會有所下降。應用touch down PCR併在體繫中添加7-deaza-dGTP能夠提高擴增產物的特異性和產率,增加擴增的保真度。結論:應用優化的PCR擴增條件將所有達託黴素生物閤成基因簇分段擴增齣來,併可擴增齣長達6 kb的片段,且序列完全正確,可以進行後續拼接。
목적::탐색고GC함량DNA적PCR확증조건,위확증체탁매소생물합성기인족급병접전정기출。방법:재PCR확증체계중,사용고보진적취합매급첨가불동농도적DMSO、7-deaza-dGTP등증강제,병선택합괄적PCR순배정서,우화부함GC적DNA적PCR확증조건。결과:향반응체계중액외첨가1%~4%적DMSO가이현저제고부함GC적DNA적PCR확증산물량,단회강저기특이성;7-deaza-dGTP가이제고확증산물적특이성급보진도,단산량회유소하강。응용touch down PCR병재체계중첨가7-deaza-dGTP능구제고확증산물적특이성화산솔,증가확증적보진도。결론:응용우화적PCR확증조건장소유체탁매소생물합성기인족분단확증출래,병가확증출장체6 kb적편단,차서렬완전정학,가이진행후속병접。
Objective: To determine the optimized conditions for PCR amplification of DNA with rich GC in or-der to amplify DNA fragments from daptomycin biosynthetic gene cluster and further for assembly. Methods:DMSO and 7-deaza-dGTP were added to the PCR amplification system and amplification cycling was chosen to optimize the conditions for PCR amplification of DNA with rich GC. Results: 1%~4% DMSO greatly improved tar-get product yield during PCR amplification but the specificity was reduced. 7-deaza-dGTP improved target prod-uct specificity and facilitated subsequent sequencing of GC-rich DNA, although product yield was not increased. Combination touch down PCR with 7-deaza-dGTP had good effect on PCR amplification, and will allow for the production of a wide variety of GC-rich gene constructs. Conclusion: Using this protocol, all the 1 kb DNA frag-ments from daptomycin biosynthetic gene cluster were successfully amplified and long DNA fragments up to 6 kb were also amplified, which will facilitate our thorough understanding to genes and their regulations and functions.