The comparison of the effectiveness of a modified conformation sensitive gel electrophoresis with denaturing high performance liquid chromatography

Iran Biomed J. 2008 Apr;12(2):109-14.

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: PCR; Polymorphism; Single nucleotide polymorphism; Conformation sensitive gel electrophoresis; Denaturing high performance liquid chromatography.

Publication types

  • Comparative Study

MeSH terms

  • Base Sequence / genetics
  • Chromatography, High Pressure Liquid / methods
  • DNA / chemistry*
  • DNA / genetics*
  • DNA Mutational Analysis
  • Electrophoresis, Agar Gel / methods
  • Exons / genetics
  • Humans
  • Membrane Proteins / genetics
  • Nucleic Acid Conformation*
  • Nucleic Acid Denaturation*
  • Phosphoric Monoester Hydrolases / genetics
  • Point Mutation / genetics

Substances

  • Membrane Proteins
  • DNA
  • SGPP2 protein, human
  • Phosphoric Monoester Hydrolases