The comparison of the effectiveness of a modified conformation sensitive gel electrophoresis with denaturing high performance liquid chromatography.
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.
Khorram Khorshid
Hamid Reza
HR
Genetic Research Centre, the Social Welfare and Rehabilitation Sciences University, Tehran, Iran.|Dept.  of Genetics, University of Leicester, Leicester, UK.
Dalgleish
Raymond
R
Dept.  of Genetics, University of Leicester, Leicester, UK.
D001483:Base Sequence / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004252:DNA Mutational Analysis; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005091:Exons / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D017354:Point Mutation / Q000235:genetics
D008565:Membrane Proteins; D004247:DNA; C498526:SGPP2 protein, human; D010744:Phosphoric Monoester Hydrolases