Rapid detection of non-deletional alpha-thalassemia mutations by PCR-LIS-SSCP

Objective: To establish a rapid and convenient single strand conformation polymorphism (SSCP) analysis method for detecting the point mutation of non-deletional alpha-thalassemia.

Methods: The 543bp DNA fragment spanning the hot spot region mainly responsible for non-deletional alpha-thalassemia was nested amplified using the selective amplification of alpha2 globin gene as a template and was denatured with low ionic strength(LIS) solution followed by SSCP analysis.

Results: LIS buffer was more efficient for ssDNA formation than formamide buffer was and the formation of ssDNA was very stable. In addition to a normal electrophoresis pattern, at least three SSCP profiles can be detected by the present method when the DNA samples bearing non-deletional genes of Hb H disease were screened. Confirmed by DNA sequencing analysis, the DNAs represented these profiles have turned out to be the different three mutants, i.e., the alphaCS mutation, the alpha QS mutation and the alphaWestmead mutation, respectively. Only 3 hours were needed to complete the electrophoresis procedure of this method.

Conclusion: PCR-LIS-SSCP can be used as a tool in rapid screening for the alterations in human alpha-globin gene.