Khorram Khorshid Hamid Reza, Dalgleish Raymond
Genetic Research Centre, the Social Welfare and Rehabilitation Sciences University, Tehran, Iran.
Dept. of Genetics, University of Leicester, Leicester, UK.
Iran Biomed J. 2008 Apr;12(2):109-14.
Several methods have been developed for detection of sequence variation in genes and each has its advantages and disadvantages. A disadvantage of them is that the simpler, cost-effective methods are commonly perceived as being less sensitive in their detection of sequence variation, whereas those with proven sensitivity have a requirement for complex or expensive laboratory equipment. In this context, we undertook improvements to the conformation sensitive gel electrophoresis (CSGE) method which provides a cost-effective approach to mutation detection and compared the results with scanning carried out using denaturing high performance liquid chromatography (DHPLC) which utilises a dedicated analyser.
We designed PCR primers to amplify the seven protein-coding exons of the human SPP2 gene which encodes secreted phosphoprotein 24 (spp24) such that the amplified products included the immediately-adjacent intronic regions. Five improvements were made to the CSGE method that was used to the scan the PCR-amplified DNA. The scanning was then repeated using DHPLC and the results were compared.
Using CSGE, a single nucleotide polymorphism was discovered in exon 2 and another in intron 2 of the gene. Re-scanning of the same regions by DHPLC detected no additional sequence polymorphisms.
With modification of the original protocol, CSGE is capable of providing a simple and cost-effective approach to the detection of DNA sequence polymorphisms that appears to be comparable in sensitivity to DHPLC.
已开发出多种检测基因序列变异的方法，每种方法都有其优缺点。这些方法的一个缺点是，较简单、成本效益高的方法通常被认为在检测序列变异时灵敏度较低，而那些已证实具有高灵敏度的方法则需要复杂或昂贵的实验室设备。在此背景下，我们对构象敏感凝胶电泳（CSGE）方法进行了改进，该方法为突变检测提供了一种经济有效的途径，并将结果与使用变性高效液相色谱（DHPLC）进行的扫描结果进行了比较，DHPLC使用了专用分析仪。
我们设计了PCR引物，以扩增编码分泌磷蛋白24（spp24）的人类SPP2基因的七个蛋白质编码外显子，使得扩增产物包括紧邻的内含子区域。对用于扫描PCR扩增DNA的CSGE方法进行了五项改进。然后使用DHPLC重复扫描，并比较结果。
使用CSGE，在该基因的外显子2和内含子2中分别发现了一个单核苷酸多态性。通过DHPLC对相同区域进行重新扫描，未检测到其他序列多态性。
通过对原始方案的修改，CSGE能够提供一种简单且经济有效的DNA序列多态性检测方法，其灵敏度似乎与DHPLC相当。
