Optimization of the single‐strand conformation polymorphism (SSCP) technique for detection of point mutations
Optimization of the single‐strand conformation polymorphism (SSCP) technique for detection of point mutations
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用于检测点突变的单链构象多态性（SSCP）技术的优化
作者：
D. Glavač;M. Dean
在小鼠β珠蛋白基因的193 bp长的DNA片段中，对86个随机分布的点突变进行了单链构象多态性（SSCP）分析，以检测单碱基取代的效率。改变多个参数，包括电泳温度、缓冲液浓度、凝胶浓度、丙烯酰胺与双丙烯酰胺的比例和/或向凝胶基质中添加不同化合物。具有较高浓度丙烯酰胺和较低交联的凝胶给出了最佳分离，并且在4°C下，在5%或7.5%（2.6%C）丙烯酰胺凝胶上，所有86个突变都可以与野生型清楚地区分。大多数突变也可在含有5%尿素或甲酰胺或10%二甲亚砜或蔗糖的凝胶上从野生型中分辨出来。通过不对称PCR-SSCP技术跟踪富含嘌呤和嘧啶的单链的相对位置。我们发现，大部分的信息来自嘌呤丰富的链，这似乎是更敏感的凝胶中的变化。突变的位置或类型与其被检测的能力无关。然而，突变周围的相邻碱基序列似乎对迁移率有影响。例如，GC富集区的A→G置换显著增加了嘌呤富集链的迁移率变化，而大多数G→A置换则降低了迁移率变化。我们得出结论，如果针对特定DNA片段优化影响分离的参数，SSCP是检测点突变的非常有效的方法。© 1993 Wiley利斯公司
The efficiency of detection of single base substitutions by single‐stranded conformation polymorphism (SSCP) analysis was tested on 86 randomly distributed point mutations in a 193‐bp‐long DNA fragment of the mouse β‐globin gene. Multiple parameters were varied, including electrophoresis temperature, buffer concentration, gel concentration, acrylamide‐to‐bis‐acrylamide ratio, and/or addition of different compounds to the gel matrix. Gels with a higher concentration of acrylamide and lower crosslinking gave optimal separation, and all 86 mutations can be clearly distinguished from the wild type on a 5% or 7.5% (2.6% C) acrylamide gel at 4°C. Most of the mutations are also resolvable from wild type on gels with 5% urea or formamide, or 10% dimethylsulfoxide or sucrose. The relative position of the purine and pyrimidine‐rich single strands were followed by an asymmetric PCR–SSCP technique. We found that most of the informativity comes from the purine rich strand, which appears to be much more sensitive to changes in the gel. The position or type of mutation showed no correlation with its ability to be detected. However, the neighboring base sequence around the mutation appears to have an effect on mobility. For example, A→G substitutions in GC‐rich regions significantly increase the mobility shift of the purine‐rich strand, while most G→A changes decrease it. We conclude that SSCP is a very efficient method for the detection of point mutations, if the parameters that effect the separation are optimized for a particular DNA fragment. © 1993 Wiley‐Liss, Inc.
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DOI：
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