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

基因突变与遗传性疾病和癌症的关系已经被研究，DNA单碱基突变的检测应该有助于对这些疾病的医学诊断。单链构象多态(SSCP)和限制性片段长度多态(RFLP)等方法已被用于检测点突变和单核苷酸多态(SNPs)，但这些方法仍然费时费力。在这项研究中，我们建立了一种亲和毛细管电泳法，用于DNA-聚丙烯酰胺偶联物的单碱基差示检测寡核苷酸。以甲基丙烯酰氧基丁二酰亚胺为原料，与5‘端带有氨基己基键的寡核苷酸发生偶联反应，合成了DNA-聚丙烯酰胺偶联物。注入毛细管中的DNA-聚丙烯酰胺偶联物可以作为假固定化亲和配体，因为它们在迁移率方面与非离子聚丙烯酰胺相似。这种新型的亲和毛细管电泳法成功地在30min内分离了含有癌基因(K-ras)序列的60mer寡聚脱氧核糖核苷酸和带有单碱基取代的版本。结论：利用DNA-PolyAAm偶联物作为亲和配基，可以在毛细管电泳法上分离出单碱基差异的DNA。该分析体系具有良好的应用前景，可用于新型基因突变检测。

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.