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Updated: Aug 2, 2026
Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
Published on: March 29, 2017
[Rapid detection of non-deletional alpha-thalassemia mutations by PCR-LIS-SSCP]
1Institute of Molecular Biology, The First Military Medical University, Guangzhou, 510515, P. R. China. gzxuxm@publicl.guangzhou. gd.cn
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 alpha&supCS; mutation, the alpha &supQS; mutation and the alpha&supWestmead; 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.
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