[Structural analysis of abnormal hemoglobin by the polymerase chain reaction (PCR) of genomic DNA].
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.
Harano
T
T
Department of Biochemistry, Kawasaki Medical School, Kurashiki.
Harano
K
K
Kushida
Y
Y
Ueda
S
S
D004247:DNA / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D005796:Genes; D006455:Hemoglobins, Abnormal / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction
D006455:Hemoglobins, Abnormal; D004247:DNA