CAJ | 학술논문

背景:真菌DNA的提取在真菌基因工程以及分子生物学研究中占有重要地位.DNA的提取效率,尤其是DNA的质量严重影响实验结果.目的:建立提取病原真菌基因组DNA的方法,探讨简单重复序列-PCR反应体系中各成分的最佳组合.设计、时间及地点:DNA提取方法对照分析及正交实验,于2004-06,2006-12在吉林大学白求恩医学院病原生物学教研室真菌学研究室进行.材料:将接种临床标本的马铃薯葡萄糖液体培养基、马铃薯葡萄糖琼脂培养基、酵母蛋白胨液体培养基28℃培养3-7 d后,选取可疑菌落进行分离、纯化.方法:比较玻璃珠-盐析法、CTAB法、GeneTLE~(TM)抽提法3种不同DNA提取方法对菌体DNA质量的影响;利用正交设计,选择Taq DNA聚合酶、模板DNA、dNTP、引物4个因素,在3个水平上根据L9(3~4)正交表进行试验,对真菌简单重复序列-PCR(SSR-PCR)体系进行优化分析;并通过PCR选择适宜的退火温度及循环次数.主要观察指标:根据扩增获得带型的多态性和特异性,确定最佳反应体系.结果:Gene TLE~(TM)抽提法全部在相应的PCR体系中扩增出目的片段,且带型清楚度、明亮度优于其余两种方法.SSR-PCR反应体系正交试验结果显示,根据尺值大小确定因素显著性顺序为模板DNA(2.67)>Taq DNA聚合酶(2.00)>dNTP(0.67)>引物(0.33);根据ki值大小确定各因素优化水平组合为:模板DNA 30 mg/L,Taq DNA聚合酶1 U,dNTP150 μmol/L,引物0.5 μmol/L.但由于引物作为影响因素的R值小,是非显著因素,因此引物取A水平即0.25 μmol/L.反应条件以53℃退火温度、35个循环次数为最佳.结论:Gene TLE~(TM)提取法提取DNA的效率更高、操作步骤快速简单.根据正交试验的优化结果,SSR-PCR最佳反应体系为模板DNA 30 mg/L,Taq DNA聚合酶1 U,dNTP 150 μmol/L,引物0.25 μmol/L.反应条件以53℃退火温度、35个循环次数为最佳.
배경:진균DNA적제취재진균기인공정이급분자생물학연구중점유중요지위.DNA적제취효솔,우기시DNA적질량엄중영향실험결과.목적:건립제취병원진균기인조DNA적방법,탐토간단중복서렬-PCR반응체계중각성분적최가조합.설계、시간급지점:DNA제취방법대조분석급정교실험,우2004-06,2006-12재길림대학백구은의학원병원생물학교연실진균학연구실진행.재료:장접충림상표본적마령서포도당액체배양기、마령서포도당경지배양기、효모단백동액체배양기28℃배양3-7 d후,선취가의균락진행분리、순화.방법:비교파리주-염석법、CTAB법、GeneTLE~(TM)추제법3충불동DNA제취방법대균체DNA질량적영향;이용정교설계,선택Taq DNA취합매、모판DNA、dNTP、인물4개인소,재3개수평상근거L9(3~4)정교표진행시험,대진균간단중복서렬-PCR(SSR-PCR)체계진행우화분석;병통과PCR선택괄의적퇴화온도급순배차수.주요관찰지표:근거확증획득대형적다태성화특이성,학정최가반응체계.결과:Gene TLE~(TM)추제법전부재상응적PCR체계중확증출목적편단,차대형청초도、명량도우우기여량충방법.SSR-PCR반응체계정교시험결과현시,근거척치대소학정인소현저성순서위모판DNA(2.67)>Taq DNA취합매(2.00)>dNTP(0.67)>인물(0.33);근거ki치대소학정각인소우화수평조합위:모판DNA 30 mg/L,Taq DNA취합매1 U,dNTP150 μmol/L,인물0.5 μmol/L.단유우인물작위영향인소적R치소,시비현저인소,인차인물취A수평즉0.25 μmol/L.반응조건이53℃퇴화온도、35개순배차수위최가.결론:Gene TLE~(TM)제취법제취DNA적효솔경고、조작보취쾌속간단.근거정교시험적우화결과,SSR-PCR최가반응체계위모판DNA 30 mg/L,Taq DNA취합매1 U,dNTP 150 μmol/L,인물0.25 μmol/L.반응조건이53℃퇴화온도、35개순배차수위최가.
BACKGROUND: Extraction of fungal DNA plays an important role in fungal genetic engineering and molecular biology research.The result of experiment is affected seriously by the efficiency of extracting DNA especially the quality of DNA. OBJECTIVE: To develop a method for extracting genomic DNA of pathogenic fungi and discuss the optimal combination of components in simple sequence repeat PCR (SSR-PCR) system. DESIGN, TIME AND SETTING: A comparative analysis of DNA extraction methods and an orthogonal experiment were conducted in the Mycology Research Lab of Department of Pathogenobiology in Norman Bethune College of Medicine, Jilin University from July 2004 to December 2006.MATERIALS: Clinical specimens were inoculated on Potato dextrose agar, Potato dextrose broth and Yeast extract peptone dextrose and cultivated under the temperature of 28 ℃ for 3-7 days, after which suspectable colonies were selected to be isolated and purified.METHODS: Three kinds of methods of extracting DNA(beading-salt fractionation method, CTAB method and Gene TLE~(TM) extraction method ) were compared in terms of their effects on DNA quality; Experiment was performed with orthogonal design to four factors (Taq DNA polymerase, template DNA, dNTP, primers) in three levels on the basis of L9 (3~4) orthogonal table, The appropriate annealing temperature and cycles were determined through PCR.MAIN OUTCOME MEASURES: The optimal reaction system determined according to the polymorphism and specificity of amplification of banding pattern.RESULTS: The objective fragments were all amplified by Gene TLE~(TM) extraction method, and the banding patterns obtained were clearer and brighter compared with the other two methods. The result of orthogonal experiment on SSR-PCR system showed that,according to the value of R, the significance of factors followed by ascending were template DNA (2.67), Taq DNA polymerase (2.00), dNTP (0.67) and primers (0.33). According to the value of ki, the optimal level of each factor combination was 30 mg/L template DNA, 1U Taq DNA polymerase, 150 μmol/L dNTP, 0.5 μmol/L primer. However, because primers were nonsignificant factors, which was presented by their small R value, we took A level of primer as 0.25 μmol/L. The best reaction condition was 55 ℃ annealing temperature and 35 cycles.CONCLUSION: The Gene TLE~(TM) method shows higher efficiency of extracting DNA and its operation is fast and simplel According to the results of orthogonat experiment, the optimal SSR-PCR system was 30 mg/L template DNA, 1U Taq DNA polymerase, 150 μmol/L dNTP and 0.25 μmol/L primer. The best reaction condition was 55℃ annealing temperature and 35 cycles.