Harano T, Harano K, Kushida Y, Ueda S
Department of Biochemistry, Kawasaki Medical School, Kurashiki.
Rinsho Byori. 1990 Sep;38(9):1067-72.
Determination of the primary structure of abnormal Hbs on the basis of DNA sequencing of the globin gene obtained from a carrier of abnormal Hb was performed. DNA obtained from the leukocytes of the peripheral blood was amplified by the polymerase chain reaction (PCR) using the proper amplification primer set. Amplified DNA was digested with two different restriction endonucleases and cloned to vector M 13 mp 18 or mp 19, which had been digested with the same enzymes. DNA sequencing was done by the dideoxy chain termination method using T 7 DNA polymerase, and the abnormal Hbs whose primary structure was determined were as follows: Hb Fukuoka [beta 2 His(CAC/T)----Tyr(TAT)], Hb Machida [beta 6 Glu(GAG)----Gln (CAG)], Hb Hope [beta 136 Gly(GGT)----Asp(GAT)], Hb Hiroshima [beta 146 His(CAC)----Asp(GAC)] and Hb Kodaira [beta 146 His(CAC)----Gln(CAA)]. This method for determining the primary structure of abnormal Hbs might be more effective than the ordinary method, which involves amino acid analysis and amino acid sequencing of the abnormal peptide obtained from abnormal Hb.
基于从异常血红蛋白携带者获得的珠蛋白基因的DNA测序，对异常血红蛋白的一级结构进行了测定。使用合适的扩增引物对，通过聚合酶链反应（PCR）对外周血白细胞获得的DNA进行扩增。扩增的DNA用两种不同的限制性内切酶消化，并克隆到用相同酶消化过的载体M 13 mp 18或mp 19中。使用T 7 DNA聚合酶通过双脱氧链终止法进行DNA测序，已确定其一级结构的异常血红蛋白如下：福冈血红蛋白[β2组氨酸（CAC/T）→酪氨酸（TAT）]、町田血红蛋白[β6谷氨酸（GAG）→谷氨酰胺（CAG）]、希望血红蛋白[β136甘氨酸（GGT）→天冬氨酸（GAT）]、广岛血红蛋白[β146组氨酸（CAC）→天冬氨酸（GAC）]和小平血红蛋白[β146组氨酸（CAC）→谷氨酰胺（CAA）]。这种确定异常血红蛋白一级结构的方法可能比普通方法更有效，普通方法涉及对从异常血红蛋白获得的异常肽进行氨基酸分析和氨基酸测序。
