Separation of Oligodeoxyribonucleotides Having Single-base Mutation by Affinity Capillary Electrophoresis Using a DNA- polyacrylamide Conjugate

基因突变已经被研究与遗传疾病和癌症有关，DNA单碱基突变的检测应该有助于对这些疾病进行医学诊断。单链构象多态性（SSCP）和限制性片段长度多态性（RFLP）等检测点突变和单核苷酸多态性（snp）的方法已经被开发出来，但它们仍然耗时且费力。需要快速、简便、灵敏的检测方法。在这项研究中，我们开发了一种亲和毛细管电泳，用于使用dna -聚丙烯酰胺偶联物检测寡脱氧核糖核苷酸的单碱基差异。将甲基丙烯酰氧丁二酰亚胺与5′端有氨基己基连接的ODN偶联得到的大单体ODN与丙烯酰胺共聚合成dna -聚丙烯酰胺共轭物。将dna -聚丙烯酰胺缀合物注射到毛细管中可以作为准固定化亲和配体，因为它们在迁移速率方面的行为与非离子型聚丙烯酰胺相似。这种新型亲和毛细管电泳在30分钟内成功地分离了具有致癌基因（K-ras）序列和单碱基取代的寡脱氧核糖核苷酸（60 mer）。此外，通过PCR扩增从人类细胞中获得的样本dna可以进行该分离系统。综上所述，利用dna -聚aam缀合物作为亲和配体，可以在毛细管电泳系统中分离出单碱基差异的dna。该分析系统在新型基因突变检测中具有广阔的应用前景。

Gene mutations have been studied in relation to genetic diseases and cancers, and the detection of DNA single-base mutations should be helpful in making medical diagnoses of these disorders. A number of methods such as single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) have been developed for detecting point mutations and single nucleotide polymorphisms (SNPs), but they are still time-consuming and laborious. Rapid, facile and sensitive detection methods are required.In this study, we have developed an affinity capillary electrophoresis for a single-base difference detection of oligodeoxyribonucleotides using a DNA-polyacrylamide conjugate. The DNA-polyacrylamide conjugate was synthesized through copolymerization between acrylamide and ODN macromonomer, which was obtained by coupling reaction between methacryloyloxysuccinimide and ODN having amino-hexyl linker at its 5'-end. The DNA-polyacrylamide conjugates injected into a capillary can serve as pseudo-immobilized affinity ligands because they behave similarly to non-ionic polyacrylamide in terms of migration rate. This novel affinity capillary electrophoresis was successfully applied to separation of oligodeoxyribonucleotides (60 mer) having an oncogene (K-ras) sequence and a version with single-base substituted within 30 minutes. Moreover, sample DNAs obtained from human cells through PCR amplification can be subjected to this separation system.In conclusion, by using DNA-polyAAm conjugate as an affinity ligand we can separate DNAs having a single-base difference on capillary electrophoresis system. This analytical system is promising for the novel gene mutation assay.