J Clin Pathol Qual Control.  2001 Jun;23(1):193-199.

Development of Mutation Scanning Method for the Molecular Diagnosis of beta-thalassemia using Dideoxy Fingerprinting (ddF)

• Joo SI
• Park SS

Abstract

BACKGROUND: beta-Thalassaemia is the most common single-gene disorders due to the mutations of the beta-globin gene. Because beta-thalassemic mutations are scattered throughout the beta-globin gene, mutation scanning technique may be useful to isolate the causative mutation. Dideoxy fingerprinting (ddF) is a hybrid technique which combines aspects of single strand conformational polymorphism (SSCP) and dideoxy sequencing. We tried to establish ddF method to detect the beta-thalassemic mutations.
METHODS
We carried out ddF screening for eleven types of known beta-thalassemic mutations. We performed dideoxy termination reaction using Thermo Sequenase radiolabeled terminator cycle sequencing kit, and radioisotope labelled ddATP or ddCTP. We used the PCR product of whole beta-globin gene as templates, and designed new primers of which locations were less than 200 bp distant from each mutation. After SSCP analyses with 0.5X MDE gel and autoradiography, we evaluated ddF results in each mutation.
RESULTS
Using ddF, we could completely detect all eleven types of beta-thalassemic mutations, and classified those results into four groups. There was no significant difference between two kinds of dideoxy nucleotides (ddATP and ddCTP). Polymorphisms within the beta-globin gene were not problematic, because we used heterozygote DNA for each polymorphism as a control.
CONCLUSIONS
We developed ddF protocol to be able to detect all types of beta-thalassemic mutations found Koreans. ddF is a rapid and highly sensitive mutation scanning method, and may be applied for the mutation analyses of other genetic diseases.

MeSH Terms

Autoradiography
beta-Globins
beta-Thalassemia*
Dermatoglyphics*
Diagnosis*
DNA
Heterozygote
Mass Screening
Nucleotides
Polymerase Chain Reaction
Polymorphism, Single-Stranded Conformational
DNA
Nucleotides
beta-Globins