CN104120173B - 碱性品红及其衍生物在聚丙烯酰胺凝胶上dna检测中的应用 - Google Patents
碱性品红及其衍生物在聚丙烯酰胺凝胶上dna检测中的应用 Download PDFInfo
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
本发明涉及DNA检测技术，具体地说是碱性品红(Basic Fuchsin)及其衍生物在DNA检测中的应用。本发明还提供了应用碱性品红聚丙烯酰胺凝胶上DNA检测的方法，包括步骤：将DNA样品电泳后的凝胶在染色液中染色；显影。本发明具有灵敏度高、操作简单迅速、重现性好、线性范围广、使用安全、成本低廉等优点。
Description
技术领域
本发明涉及DNA检测技术，具体地说涉及DNA检测的一种新型染料。
背景技术
在生命科学研究领域中，作为生物体内的重要大分子，DNA是研究生命现象与本质的物质基础，特别随着基因组学、功能基因组学和结构基因组学的快速发展，它的应用范围越来越广，主要包括：对特定基因功能的识别、新药筛选、疾病诊断、药物作用机理、生物各个时期的发育变化等方面的研究，它几乎渗透到生命科学的各个领域。因此，对DNA的研究具有重要的意义。目前，聚丙烯酰胺凝胶电泳是高效分离和分析DNA分子的一种常用方法，使得凝胶上DNA的检测技术对DNA的相关研究起着至关重要的作用[1]。
测定DNA含量的方法有多种，目前用于聚丙烯酰胺凝胶上DNA研究和测定的方法有荧光染色法、染料染色法、负染法、银染法等。DNA染料染检测法因具有快速便捷、价格低廉、低毒等优点而成为DNA检测研究领域的热点之一。
碱性品红为绿色金属光泽结晶，溶于乙醇和戊醇，微溶于水，溶液呈红色，不溶于乙醚；最大吸收波长(乙醇中)543nm。到目前为止，还没有将其用于DNA检测的报道[2，3]。
发明内容
本发明的目的在于提供碱性品红及其衍生物在DNA检测中的应用。
本发明所述的碱性品红相关化合物是指以碱性品红为母核的钠盐、钾盐、铵盐等。
碱性品红母核为：
下面是本发明一个可用的凝胶检测方法，其包括步骤：
1)将经聚丙烯酰胺电泳后的DNA样品凝胶置于染色液中染色3-10min，其中所述染色液为含按重量体积比为0.01％-0.1％碱性品红水液，弃染色液；优选染色时间为5min，优选碱性品红水溶液浓度为0.04％。
2)加入洗脱液洗脱5-30s，两次，其中所述染色液为去离子水，弃洗脱液；优选洗脱时间为10s。
3)加入显影液显影1-5min，其中所述显影液为含按重量体积比为0.01-0.1％铁氰化钾(20-60℃)水溶液，弃显影液。优选显影时间为2min，优选铁氰化钾浓度为0.1％，优选温度为35-50℃。
4)加入终止液终止1-5min，其中所述终止液为20-60℃去离子水。优选时间为2min，优选温度为35-50℃。
用碱性品红作为DNA检测染料具有如下优点：
1)灵敏度高：比普通染料染色的灵敏度高很多倍；
2)操作简单迅速：操作简便，可在10min内完成；
3)重现性好：受摇床摆动频率等外部条件影响较小；
4)线性范围广：能在较大范围内对DNA进行半定量测定；
5)使用安全：采用低毒性的染料，提高实验操作的安全性，对环境污染小；
6)成本低廉。
附图说明
图1.碱性品红的化学结构；
图2.碱性品红染色法与其他DNA检测方法灵敏度的比较。DNA(pUC18DNA/MspIMarkers)载样量(每条带)如下：带1，2500；带2，1250；带3，620；带4，310；带5，160；带6，80；带7，40；带8，20；带9，10；带10，5pg
图3.碱性品红染色法与其他DNA检测方法在实际样品的比较。条带1-10为被被稀释的pMHP0质粒。
具体实施方式：
下面结合具体的实施例来进一步阐述本发明。应当理解，这些实施例仅用于说明本发明，而不能限制本发明的保护范围。
实施例1碱性品红DNA检测
图1是碱性品红的化学结构式。碱性品红的DNA染色实验采用下述步骤进行：
1)将经聚丙烯酰胺电泳后的DNA样品凝胶置于染色液中染色5min，其中所述染色液为含按重量体积比为0.04％碱性品红水液，弃染色液；
2)加入洗脱液洗脱10s，两次，其中所述染色液为去离子水，弃洗脱液；
3)加入显影液显影2min，其中所述显影液为含按重量体积比为0.1％铁氰化钾(35-50℃)水溶液，弃显影液。
4)加入终止液终止2min，其中所述终止液为35-50℃去离子水；
按照上述方法分别用碱性品红的钠盐、钾盐、铵盐进行DNA染色，结果表明用这些衍生物均能得到类似于碱性品红的检测结果。
聚丙烯酰胺凝胶参照《分子克隆实验指南》第三版相关部分进行。
实验例1碱性品红染色法与其他DNA检测方法灵敏度的比较
采用不同染色法进行染色，(A)碱性品红染色法；(B)GE公司银染法；(C)SYBR gold荧光检测法。DNA载样量(每条带)如下：带1，2500；带2，1250；带3，620；带4，310；带5，160；带6，80；带7，40；带8，20；带9，10；带10，5pg。结果如图2所示，SYBR gold荧光检测法在带6之后的DNA就已不能被检测到，而对于碱性品红染色法，在带8处清晰可见DNA条带，同时可见碱性品红染色法灵敏度与GE公司银染法相近。
其中碱性品红染色采用实施例1的方法进行，GE公司银染法及SYBR gold荧光检测法分别按下述方式进行：
GE公司银染法：
固定：125ml固定液，包含24％乙醇及0.6％苯磺酸，固定30min；
银染：125ml染色液，包含0.2％硝酸银及0.07％苯磺酸，染色30min；
水洗：125ml去离子水，洗1min；
显影：125ml显影液，包含2.5％碳酸钠，0.1％甲醛及0.002％硫代硫酸钠显影6min；
终止：125ml终止液，包含1％乙酸，5％乙酸钠及10％甘油终止30min。
SYBR gold荧光检测法：
水洗：125ml去离子水，洗1min；
染色：50ml由TBE缓冲液稀释成1倍的SYBR Gold染色液，染色30min。
参考文献：
[1]Bassam，B.J。，Gresshoff，P.M.，Nat.Protoc.2007，2，2649-2654.
[2]Gordon，C.，Van Deun，A.，Lumb，R.，Int.J.Tuberc.Lung.Dis.2009，13，130-135.
[3]Doemer，K.C.，White，B.A.，Anal.Biochem.1990，187，147-50.
Claims (1)
1.一种DNA检测方法，其包括步骤：
1)将经聚丙烯酰胺电泳后的DNA样品凝胶置于染色液中染色5min，其中所述染色液为含按重量体积比为0.04％碱性品红水溶液，弃染色液；
2)加入洗脱液洗脱10s，两次，其中所述染色液为去离子水，弃洗脱液；
3)加入显影液显影2min，其中所述显影液为含按重量体积比为0.1％铁氰化钾水溶液，弃显影液，显影温度为35-50℃；
4)加入终止液终止2min，其中所述终止液为35-50℃去离子水；
5)检测。
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