COMPARISON BETWEEN TWO ASO PROBES FOR DETECTION OF MUTATIONS IN BETA-GLOBIN GEN

ÁLVAREZ SM, GIMÉNEZ MS

Mendoza

Congreso; XVI Reunión Anual de la Sociedad de Biología de Cuyo; 1998

Sociedad de Biologia de Cuyo

In our country, the most widely used detection component of hibridization assays are the radioisotopes, especifically 32P.  On the other hand, there is an increase of the offer of nonradioactive methods.  Then we compared the detection by Dot Blot of  the mutation in codon 39 of the  globin gen in  thalassemic patients using both systems. DNA samples were obtained from  EDTA-anticoagulated peripheral blood with standards methods.  A  segment of 536 bp of the  globin gen was amplified by PCR and dot blotted onto membranes.  The DNAs were hybridized with ASO probes labeled 1) with -ATP32,using T4-Polinucleotide kinase (BioLabs); 2)with  Biotine (obtained from GIBCO BRL).  The detections were:1) by autorradiography with x-ray film;  2) by binding of an  Streptavidine-Alkaline Phosphatase conjugate, detection reagent incubation (Lumi-Phos 530) and exposure to x-ray film.  The films analysis showed:                                                                                                                                                                                                                                                                                                                                                                                                                                        Chemiluminiscent probe    Radioactive Probe Exposure time                      ++                     ++++ Risks of overexposure            -                        +++ Image Intensity                   +++                      +++ Sensitivity                           +++                      +++ Cost/determination               ++                       +++ Radioactive contamination     -                       ++++ So, we concluded that nonradioactive labeling systems should be considered as an important alternative.