Optimization of single-stranded conformation polymorphism (SSCP) analysis for screening for the estrogen receptor-alpha gene polymorphism P325P.
Since there are no theoretical models for single-stranded conformation polymorphism (SSCP) analysis, conditions for detecting specific mutation must be found experimentally. Previously, a time-consuming (22 hours) SSCP method was used for the detection of polymorphism in codon 325 (CCC to CCG; P325P) in exon 4 of estrogen receptor-alpha gene. The aim of our work was to study different gel loading buffers, additives to polyacrylamide gel, voltages, running times and temperatures of electrophoresis, in order to develop a better and faster SSCP analysis for screening of P325P polymorphism. Our results show that a low ionic strength gel loading buffer and 10% addition of glycerol to the 8% polyacrylamide gel (37:1) are essential for the good separation of mutated and wild-type single stranded conformers of exon 4. The most suitable conditions for electrophoresis were 300 V, 5 h and 22 degrees C. We concluded that a much faster SSCP analysis for sreening of P325P polymorphism of estrogen receptor-alpha gene was developed. Although our final result could be applied only to the detection of the described genetic polymorphism, we hope that the results of our study will be helpful to analysts using SSCP analysis in their mutation screening programs.
Bitenc
D
D
Faculty of Pharmacy of Ljubljana, Slovenia.
Marc
J
J
D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D047628:Estrogen Receptor alpha; D005091:Exons; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D008403:Mass Screening / Q000379:methods*; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D011960:Receptors, Estrogen / Q000235:genetics*; D013696:Temperature; D013995:Time; D013997:Time Factors
D003062:Codon; D047628:Estrogen Receptor alpha; D011960:Receptors, Estrogen