Open AccessIdentification of the four most common β‐globin gene mutations in Greek β‐thalassemic patients and carriers by PCR‐SSCP: advantages and limitations of the method
Author(s) -
Kakavas Konstantinos V.,
Noulas Argiris,
Chalkias Christos,
Hadjichristodoulou Christos,
Georgiou Ioannis,
Georgatsou Elena,
Bonanou Sophia
Publication year - 2006
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.20091
Subject(s) - single strand conformation polymorphism , microbiology and biotechnology , biology , gene , genetics , polymerase chain reaction , mutation , silver stain , population , dna , gene mutation , globin , medicine , environmental health
In the present study we investigated whether the single‐strand conformational polymorphism (SSCP) method could be employed to identify (rather than simply detect) the four most common β‐globin gene mutations in the Greek population: IVS‐I‐110, Cd39, IVS‐I‐1, and IVS‐I‐6. Using DNA from 50 β‐thalassemic patients and carriers, we amplified by PCR the appropriate 238‐bp region of the human β‐globin gene, analyzed the reaction products by nondenaturing polyacrylamide gel electrophoresis, and visualized the bands by silver staining. Single‐stranded DNA (ssDNA) fragments showed a reproducible pattern of bands that was characteristic of the mutations present. With the use of control samples containing six of the 10 possible combinations of the four most common β‐globin gene mutations, we were able to predict the mutations present in a quarter of the patients studied. Our predictions were confirmed independently by the amplification refractory mutation system (ARMS) method. We conclude that this non‐radioactive PCR‐SSCP method can be used to reliably identify mutations in patients, provided that suitable controls are available. Moreover, the method is easy to apply to the identification of mutations in carriers, which makes it particularly useful for population screening. J. Clin. Lab. Anal. 20:1–7, 2006. © 2006 Wiley‐Liss, Inc.