Genotyping of single nucleotide polymorphism in MDM2 genes by universal fluorescence primer PCR and capillary electrophoresis.
Single nucleotide polymorphism (SNP) 309 in the promoter region of the murine double minute 2 (MDM2) gene plays an important role in human tumorigenesis. We established a simple and effective CE method for SNP detection in the MDM2 gene. We designed one universal fluorescence-based nonhuman-sequence primer and one fragment-oriented primer, which were combined in one tube, and proceeded with the polymerase chain reaction (PCR). The amplicons were analyzed by capillary electrophoresis using single-strand conformation polymorphism method. PCR fragments generated from this two-in-one PCR displayed either T/T or G/G homozygosity or T/G heterozygosity. A total of 304 samples were blindly genotyped using this developed method, which included the DNA from 138 healthy volunteers, 43 chronic myeloid leukemia (CML) patients, and 123 colorectal cancer (CRC) patients. The results were confirmed by DNA sequencing and showed good agreement. The SSCP-CE method was feasible for SNP screening of MDM2 in large populations.
Chen
Yen-Ling
YL
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 100, Shi-chuan 1st Rd., Kaohsiung 807, Taiwan.
Chang
Ya-Sian
YS
Chang
Jan-Gowth
JG
Wu
Shou-Mei
SM
D001483:Base Sequence; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000235:genetics; D017931:DNA Primers / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D005838:Genotype; D006262:Health; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics*; D013696:Temperature
D017931:DNA Primers; D005456:Fluorescent Dyes; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2