An improved method for sequencing double stranded plasmid DNA from minipreps using DMSO and modified template preparation.
An improved method for sequencing double stranded plasmid DNA from minipreps using DMSO and modified template preparation.
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DOI：
10.1093/nar/18.19.5905
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发表时间：
1990-10
影响因子：
14.9
通讯作者：
Donald Seto
中科院分区：
文献类型：
--
作者：
Donald Seto
通过双脱氧链终止法的DNA测序可以对双链DNA模板进行，其数量和纯度由微型制备程序获得(1)。根据用于分离DNA的程序的不同，测序反应可能会受到高背景和条带模式中的伪影的影响，特别是测序凝胶上跨两条或更多条通道的条带的存在。二甲基亚砜(DMSO)已用于聚合酶链式反应(PCR)产物的测序(2)。本文描述了一种用于分离和处理质粒DNA的改良方法和一种用于使用DMSO修饰的双脱氧链末端测序反应以克服这种伪影以给出明确的DNA序列测定的方案的程序。在pOTSNcol2(3)中携带酵母热休克因子基因yHSF(3‘A54和3’A63)的E.coliDH5a新鲜生长到对数末期：过夜培养物稀释，重新生长，一天收获。用改良的Holmes和Quigley方法处理1.5ml的5ml培养物(4)。该微型制备程序包括以下步骤：1)将细菌小球重新悬浮在300/tl Stet(8%蔗糖，5%Triton X-100，50 mM EDTA，50 mM Tris-Cl，pH 8.0)和20/tl 10 mg/ml溶菌酶中；2)在室温下孵育5分钟；3)煮2分钟；4)离心并丢弃小球；5)用500/tl 2.5M醋酸铵-75%异丙醇在室温下沉淀20分钟。最后的颗粒再悬浮在50/tl的H2O中。为了确保双链模板的完全分离，采用了两个变性步骤。每50个/tl mini prep DNA，加入4/tl 2N NaOH和4/tl 2 mM EDTA，在90℃孵育5分钟。在此基础上加入6/tl 3M醋酸钠(PH 5)，14/tl水和160/tl乙醇。DNA在室温下沉淀至少10分钟。样品在室温下离心20min，用70%乙醇洗涤两次，真空干燥。将颗粒溶解在25/tl H2O中；其中，7/tl与2/tl 5x序列缓冲液(50 mM Tris-Cl，pH 7.5，60 mM NaC l，35 mM MgCl2，5 mM DTT)、1/tl DMSO和1/tl(2.9pmole)引发剂一起在65°C加热5分钟，并在干冰中快速冷冻。在快速解冻和将退火样品等分成单独的G、A、C和T反应管后，随后的DNA测序反应类似于Koop等人描述的方案。(5)标记和终止步骤均加入10%v/v DMSO(终浓度)。美国生化公司(克利夫兰，俄亥俄州)序列酶试剂盒也可以与Miniprep DNA模板和DMSO包涵体一起使用。图1显示了m
DNA sequencing by the dideoxy chain termination method can be performed on double stranded DNA templates from quantities and purities achieved with miniprep procedures (1). Depending on the procedure used to isolate the DNA, sequencing reactions can be subject to high backgrounds and artifacts in band patterns, particularly presence of bands across two or more lanes on the sequencing gel. Dimethyl sulphoxide (DMSO) has been used in sequencing polymerase chain reaction (PCR) products (2). Described herein is a procedure coupling a modified method for isolating and treating plasmid DNA and a protocol for using a DMSO-modified dideoxy chain termination sequencing reaction to overcome such artifacts to give unambiguous DNA sequence determination. E. coli DH5a bearing the yeast heat shock factor gene yHSF (3'A54 and 3'A63) in plasmid pOTSNcol2 (3) was freshly grown to late log phase: overnight cultures were diluted, re-grown, and harvested in a single day. 1.5 ml of this 5 ml culture was treated using a modified procedure of Holmes and Quigley (4). This miniprep protocol consists of the following steps: 1) resuspending the bacterial pellet in 300 /tl STET (8% sucrose, 5% Triton X-100, 50 mM EDTA, 50 mM Tris-Cl, pH 8.0) and 20 /tl 10 mg/ml lysozyme; 2) incubating at room temperature for 5 minutes; 3) boiling this suspension for 2 minutes; 4) centrifuging and discarding the pellet; and 5) precipitating with 500/tl 2.5 M ammonium acetate—75% isopropanol at room temperature for 20 minutes. This final pellet was resuspended in 50 /tl of H2O. To ensure complete double stranded template separation, two denaturation steps were utilized. Per 50 /tl miniprep DNA, 4 /tl 2 N NaOH and 4 /tl 2 mM EDTA was added, and the sample incubated at 90°C for 5 minutes. To this was added 6 /tl 3 M NaAcetate (pH 5), 14 /tl H2O and 160 /tl ethanol. The DNA was precipitated at room temperature for at least 10 minutes. Samples were centrifuged for 20 minutes at room temperature and the DNA pellet was washed twice with 70% ethanol and vacuum dried. The pellet was dissolved in 25 /tl H2O; of this, 7 /tl was heated along with 2 /tl 5x sequencing buffer (50 mM Tris-Cl, pH 7.5, 60 mM NaCl, 35 mM MgCl2, 5 mM DTT), 1 /tl DMSO and 1 /tl (2.9 pmole) primer, at 65°C for 5 minutes and quickly frozen in dry ice. After quick thawing and aliquoting the annealed sample into individual G,A, C and T reaction tubes, subsequent DNA sequencing reactions similar to protocols described by Koop et al. (5) were performed with the modification of adding 10% v/v DMSO (final concentration) to both the labeling and terminating steps. The US Biochemical Corp. (Cleveland, OH) Sequenase kit may also be used with miniprep DNA template and DMSO inclusion. Figure 1 shows m