电泳系统 Electrophoresis System——Dcode突变检测电泳系统
系统功能灵活，适合多种突变检测方法
在癌症研究发展及多种单基因或多基因疾病的研究过程中，探索未知突变起着非常重要的作用。DNA 多态性 是物种的生态与进化研究非常有用的工具。应用范围包括物种鉴定、种群结构分析及遗传多样性研究等。
DCode 系统由带温度控制模块（包括微处理器控制加热）的电泳设备、一个缓冲液再循环泵、一个搅拌器及 铺制特殊应用凝胶的附件组成。冷却槽及一个外部实验室冷却器可把运行温度控制在室温下。一块凝胶能在2 个小 时内运行64 个样品，并提供5-70°C 范围内的精确温度控制。它可与下列的电泳技术配合使用来检测单碱基变化：
- 单链构象多态性（SSCP）
- 变性梯度凝胶电泳（DGGE）
- 时间温度梯度电泳（TTGE）
- 均一变性凝胶电泳（CDGE）
为了获得最高的通量和效率，许多实验室采用多种技术。在快节奏研究环境中，不同方法间的快速切换和转换 到新技术的能力是非常关键的。DCode 系统能迎合所有主要突变检测技术的需要。其灵活的功能与优点包括：
- 温度控制选择
- 模块式设计，可满足用户现在与将来的需要
- 专用的组合配件，简化启动过程
- 针对特定技术的试剂和对照物，适合DGGE、CDGE、SSCP 和TTGE 等技术
运用DGGE搜寻未知突变
DGGE 是基于以下原理，即增加聚丙烯酰胺凝胶内变性剂的浓度会熔解双链DNA 的不同区域。当温度达到最低熔解度区域的熔解温度(Tm) 时，DNA 部分熔解，在凝胶中形成迁移率下降的分支分子（Myers et al.1987）。在均一的运行温度（50—65°C）和尿素与甲酰胺线性变性梯度条件下即形成了变性环境。梯度以垂直或平行于电泳方向形成。DGGE 是最灵敏的突变检测方法，其效率可达99%（Grompe 1993）。DCode 系统通过以下途径优化DGGE：
- 温度控制模块提供45–70°C 内的一致运行温度
- 系统可运行2 块16 x 16 cm 凝胶或4 块7.5 x 10 cm 凝胶
- 可选择的MacMelt 或WinMelt 软件优化了GC 夹及引物的置放
DGGE 的两种模式。A ，凝胶的变性梯度方向与电泳方向垂直。从原生乳腺癌种扩增的p53 基因6 号外显子的野生型和突变型等位基因通过垂直DGGE（80V，56°C，2 小时）分离。感谢AL Borresen, Radium Hospital, Oslo, Norway 提供的数据。B ，凝胶的变性梯度方向与电泳方向平行。p53 基因8 号外显子的野生型和突变型等位基因在8%丙烯酰胺：甲叉(37.5：1) 平行梯度（40–65% 变性剂）凝胶上，1 x TAE 缓冲液， 60°C，150 V，电泳2.5 个小时。左泳道，突变型等位基因；中，野生型等位基因；右，突变型与野生型等位基因。
DGGE分离示意图。所示为一个变性梯度方向与电泳方向垂直的凝胶，以及一个有单个熔解区域的目标序列。野生型和突变型样品加入到制备孔内。在低变性剂浓度时，DNA 片段为双链，但当浓度增加时双链DNA 开始熔解，形成分枝分子。到浓度非常高时，DNA 片段可熔解形成2条单链。
运用TTGE
TTGE 采用DGGE 的原理但不使用化学变性剂梯度，操作更简单、 快速并便于重复（Yoshino et al.1991）。DNA 上样于含尿素的聚丙烯酰 胺凝胶。电泳过程中逐步、均一地升高温度，从而在电泳运行过程中 产生一个线性的温度梯度。变性环境由凝胶中均一的尿素浓度加上时 间温度梯度形成。DCode 系统使TTGE 简化为简单而可重复的实践。
- 温度控制器与加热器可使温度缓慢地以0.1°C/hr 的速度上升
- 无需化学梯度，使设置和制胶更方便
- MacMelt 或WinMelt 软件帮助确定最佳温度范围
- 对照试剂与已证明有效的规程使你可以立即开展实验
TTGE 分离囊性纤维化基因(外显子7)的突变型和野生型等位基因。 使用50–60°C 的温度梯度和2°C/hr 的斜率，在130V, 1.25 x TAE 缓冲液，6M 尿素/ 6%（37.5:1）丙烯酰胺凝胶上电泳5 小时。 泳道1，突变型等位基因1154 insTC；泳道2，突变型等位基因 G330X；泳道3，突变型等位基因DF311；泳道4，突变型等位 基因R334W；泳道5，野生型等位基因。感谢L Silverman, Divison of Molecular Pathology, University of North Carolina School of Medicine 提供样品。
运用CDGE 快速筛选
CDGE 是高通量筛选的有效方法。CDGE 凝胶的最佳变性剂浓度由垂直梯度凝胶决定。一旦确定了特定变异的最佳变性剂浓度，便可在均一变性凝胶上筛选多个样品(Borresen et al.1991)。因此，采用 CDGE 时无须使用梯度，使得快速高通量筛选变得简单。DCode 系统提供：
- 简化的规程，用于优化变性剂浓度
- 已经优化了的电泳试剂和对照物，确保高通量筛选的出色结果
- 方便的16 x 16 cm 和16 x 10 cm 格式及无需胶带的制胶，简化并加速了工作流程
CDGE分离beta-球蛋白基因的突变型和野生型等位基因。 含45% 变性剂的凝胶在56°C电泳。泳道1，突变样品混合物 IVS1-1/IVS1-6；泳道2，突变样品IVS1-1；泳道3，突变样品 IVS1-6；泳道4，突变样品IVS1-110；泳道5，野生型DNA。有 关这个实验的详情，见资料＃2107。
SSCP 提供一致的结果
SSCP 因其简便性而被广泛地用于突变筛选。对一个特定样品， 我们无法预测其实验条件，因此为确保最高的灵敏度，优化凝胶电泳条件（包括运行温度）显得非常重要（Orita et al.1989, Ravnik–Glavac et al.1994）。Bio–Rad 的温度控制冷却槽保证提供可重复的运行温度，以产生一致性的结果。
- 电泳冷却槽带有陶瓷冷却棒，与标准外接的再循环冷却器相连
- 控温模块带有搅拌棒、加热器和缓冲液再循环泵，维持5–25°C 之间 的均一温度
- DCode 系统也可放置在低于室温的冷库，来代替冷却槽，
- 可容纳16 x 16 cm 和16 x 20 cm 大小的凝胶，简化了银染或荧光染色的检测
SSCP 分离p53 基因(外显子8) 的突变型和野生型等位基因。在30W 恒功率，8°C，1 x TBE 缓冲液，含3.5% 甘油的8% （37.5:1）丙烯酰胺凝胶上电泳3.5 个小时。泳道1，未变性突变型等位基因；泳道2，突变型等位基因；泳道3，野生型等位基 因；泳道4，未变性野生型等位基因。
| 目录 # | 描述 |
| DCode Systems | |
| 170-9080 | DCode System for DGGE, 120 V, for 16 cm gels with single prep well (1 mm), includes comb gasket, 2 sets of clamps, Model 475 gradient former, all parts required to cast gradient gels |
| 170-9081 | DCode System for DGGE, 220/240 V, for 16 cm gels with single prep well (1 mm), includes comb gasket, 2 sets of clamps, Model 475 gradient former, all parts required to cast gradient gels |
| 170-9082 | DCode System for DGGE, 100 V, for 16 cm gels with single prep well (1 mm), includes comb gasket, 2 sets of clamps, Model 475 gradient former, all parts required to cast gradient gels |
| 170-9083 | DCode System for DGGE, 120 V, for 10 cm gels with 2 prep wells (1 mm), includes comb gasket, 2 sets of clamps, Model 475 gradient former, all parts required to cast gradient gels |
| 170-9084 | DCode System for DGGE, 220/240 V, for 10 cm gels with 2 prep wells (1 mm), includes comb gasket, 2 sets of clamps, Model 475 gradient former, all parts required to cast gradient gels |
| 170-9085 | DCode System for DGGE, 100 V, for 10 cm gels with 2 prep wells (1 mm), includes comb gasket, 2 sets of clamps, Model 475 gradient former, all parts required to cast gradient gels |
| 170-9086 | DCode System for CDGE, 120 V, for 16 cm gels with 20 wells (1 mm), includes comb gasket, 2 sets of clamps, Model 475 gradient former, all parts required to cast gradient gels |
| 170-9089 | DCode System for TTGE, 120 V, for 16 cm gels with 20 wells (1 mm), includes comb gasket, 2 sets of clamps, Model 475 gradient former, all parts required to cast gradient gels, control reagents for TTGE |
| 170-9090 | DCode System for TTGE, 220/240 V |
| 170-9091 | DCode System for TTGE, 100 V |
| 170-9092 | DCode System for SSCP, 120 V, for 20 cm gels with 20 wells (0.75 mm), includes comb gasket, 2 sets of clamps, Model 475 gradient former, all parts required to cast gradient gels, cooling tank adaptor for use with external cooling bath, control reagents for SSCP |
| 170-9093 | DCode System for SSCP, 220/240 V |
| 170-9094 | DCode System for SSCP, 100 V |
| 170-9102 | Complete DCode System, 120 V, Mac, includes electrophoresis/temperature control module with standard and cooling tanks, sandwich core, Model 475 gradient former with all accessories required to cast gradient gels, WinMelt software, control reagents for DGGE, CDGE, TTGE and SSCP systems |
| 170-9103 | Complete DCode System, 220/240 V, Mac, includes electrophoresis/temperature control module with standard and cooling tanks, sandwich core, Model 475 gradient former with all accessories required to cast gradient gels, WinMelt software, control reagents for DGGE, CDGE, TTGE and SSCP, plates, combs and spacers to cast 1 mm and 0.75 mm thick 10, 16, and 20 cm gels as described for the DGGE, CDGE, TTGE, and SSCP systems |
| 170-9104 | Complete DCode System, 100 V, Mac, includes electrophoresis/temperature control module with standard and cooling tanks, sandwich core, Model 475 gradient former with all accessories required to cast gradient gels, WinMelt software, control reagents for DGGE, CDGE, TTGE and SSCP systems |
| 170-9105 | Complete DCode System, 120 V, PC, includes electrophoresis/temperature control module with standard and cooling tanks, sandwich core, Model 475 gradient former with all accessories required to cast gradient gels, WinMelt software, control reagents for DGGE, CDGE, TTGE and SSCP, plates, combs and spacers to cast 1 mm and 0.75 mm thick 10, 16 and 20 cm gels as described for the DGGE, CDGE, TTGE, and SSCP systems |
| 170-9106 | Complete DCode System, 220/240 V, PC, includes electrophoresis/temperature control module with standard and cooling tanks, sandwich core, Model 475 gradient former with all accessories required to cast gradient gels, WinMelt software, control reagents for DGGE, CDGE, TTGE and SSCP, plates, combs and spacers to cast 1 mm and 0.75 mm thick 10, 16, and 20 cm gels as described for the DGGE, CDGE, TTGE, and SSCP systems |
| 170-9107 | Complete DCode System, 100 V, PC, includes electrophoresis/temperature control module with standard and cooling tanks, sandwich core, Model 475 gradient former with all accessories required to cast gradient gels, WinMelt software, control reagents for DGGE, CDGE, TTGE and SSCP, plates, combs and spacers to cast 1 mm and 0.75 mm thick 10, 16, and 20 cm gels as described for the DGGE, CDGE, TTGE, and SSCP systems |
| DCode Adaptor Kits | |
| 170-9125 | DGGE Kit, for 16 cm gels with single prep well (1 mm), includes sandwich clamps, pressure clamp, comb gasket and holder, fittings required for gradient gel casting |
| 170-9126 | DGGE Kit, for 10 cm gels with 2 prep wells (1 mm), includes sandwich clamps, pressure clamp, comb gasket and holder, fittings required for gradient gel casting |
| 170-9127 | CDGE/TTGE Kit, for 16 cm gels with 20 wells (1 mm), includes sandwich clamps, pressure clamp, comb gasket and holder, fittings required for gradient gel casting |
| 170-9128 | Complete SSCP Kit, for 20 cm gels with 20 wells (0.75 mm), includes sandwich clamps, cooling finger adaptor for use with external chiller |
| 170-9129 | Basic SSCP Kit, for 20 cm gels with 20 wells (0.75 mm), includes sandwich clamps |
| DCode Accessories | |
| 170-9034 | MacMelt Software, Mac |
| 170-9042 | Model 475 Gradient Delivery System, includes cam-operated manual gradient former, 2 each of 10 and 30 ml syringes, all accessories required to cast gradient gels |
| 170-9140 | Electrophoresis Cooling Tank, with cooling adaptor for hook-up to laboratory recirculating chiller |
| 170-9240 | WinMelt Software, PC/Windows |
| Electrophoresis Reagents and DNA Control Reagents | |
| 170-9150 | DCode Control Reagent Kit for DGGE/CDGE/TTGE, includes primers (one GC-clamped) and DNA templates for production of wild-type and mutant DNA |
| 170-9151 | DCode Control Reagent Kit for SSCP, includes primers and DNA templates for production of wild-type and mutant DNA |
| 170-9170 | DCode Electrophoresis Reagent Kit for DGGE, includes 500 ml 40% acrylamide/bis (37.5:1), 2 x 1 L 50x TAE buffer, 225 ml 100% deionized formamide, 10 ml 10 mg/ml ethidium bromide, 10 ml DCode dye solution, 5 ml TEMED, 1 ml 2x gel loading dye, 10 g ammonium persulfate |
| 170-9172 | DCode Electrophoresis Reagent Kit for SSCP, includes 500 ml 40% acrylamide solution, 500 ml 2% bis-acrylamide solution, 100 ml glycerol, 6 x 1 L 10x TBE buffer, 2x SSCP gel loading dye, 5 ml TEMED, 10 g ammonium persulfate |
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