生物素标记寡核苷酸探针探测扩增的病理性突变珠蛋白基因

用末端转移酶催化生物素核苷酸底物(Biotin-ll-dUTP)共价连接在合成的寡核苷酸3’羟基末端,从而合成了两种寡核苷酸探针(β~T_(41-42)及β~A_(41-42))。用它们分别与克隆化扩增的正常和突变的β—珠蛋白基因片段杂变。结果表明该探针都具有与~(32)P探针相似的特异性,其杂交的灵敏度为2—3pg(特异序列)。进而将探测HbS基因的正常和异常两种寡核苷酸19聚体(β~A_6和β~S_6)用~(32)P和生物素分别标记;将HbS杂合子病人的白细胞DNA经聚合酶链反应(PCR)法扩增,并以含正常β—珠蛋白基因的DNA片段作对照,与两种探针分别进行斑点杂交。所得结果完全一致;Hbs杂合子DNA对正常和异常探针都显出杂交信号,而正常DNA只与β~A探针显杂交信号。

Biotin-Labeled Oligonucleotide Probes for Detection of the Amplified Pathogenic Mutants of Globin Gene

Biotin-labeled probes were enzymatically prepared from allele specific 19mer oligonucleotides by the use of biotin-11-dUTP and terminal deoxynucleotidyl transferase (TdT) . Only one biotin molecule was transfered to the 3' end of a 19mer. the specificity of biotinylated probe was comparable to 32P labeled one and their hybrization sensitivity was corresponding to about 2-3pg of the related sequence in target DNA.A DNA sample from white blood cells of HbS heterozygote was amplified by polymerase chain reaction (PCR) . The amplified fragments were detected by the biotinylated 19mer probes (β6S and β6A) on dot blots, using the 32P-labeled 19mer probes as control. The hybridization signal was developed in the heterozygote DNA both with normal and mutant probes whereas the signal for the normal β-globin gene DNA was detected only with the β6A probe It showed that the biotinylated oligonucleotide probe hybridization combined with PCR would be able to detect some genetic diseases through a simple, accurate and nonradioactive dot blotting technique.