Superparamagnetic-bead Based Method: An Effective DNA Extraction from Dried Blood Spots (DBS) for Diagnostic PCR

Autor: Mahmoud Mohammed Sirdah
Jazyk: angličtina
Rok vydání: 2014
Předmět:
Zdroj: Journal of Clinical and Diagnostic Research, Vol 8, Iss 4, Pp FC01-FC04 (2014)
Druh dokumentu: article
ISSN: 2249-782X
0973-709X
DOI: 10.7860/JCDR/2014/8171.4226
Popis: Introduction: Storing blood as dried spots on filter paper is a trustworthy approach used in genetic screening issues which justifies the necessity for a reliable DNA extraction method. The present work aims to investigate the effectiveness of superparamagnetic-bead based method in extracting DNA from dried blood spots (DBS). Materials and Methods: Sixteen venous blood samples collected in K3-EDTA tubes (400µl of whole blood) were used for the spotting (4 circles each 100µl) on Ahlstrom 226 grad filter papers, for extraction and comparison. To ensure effectiveness, the extracted DNA was checked for quantity using the Quant-iT™ dsDNA Broad-Range Assay Kit and for quality by polymerase chain reaction (PCR) amplification of 344 bp segment of the HBB gene. Hybridization assays based on the dynamic allele specific hybridization (DASH) technique for two hemoglobin beta (HBB) mutations in genomic DNA extracted from DBS of ß-thalassemia patients were also performed to ensure the quality of extraction. Results: The results revealed a compatible effectiveness of the superparamagnetic-bead based method in extracting DNA from DBS particularly when incubating the DBS with lysis buffers BL+BLM overnight. A mean concentration of 21ng/ µl was obtained with lysis buffers BL+BLM overnight incubation compared to 5.2 ng/µl for 2 h incubation with lysis buffers BL+BLM and 4.7 ng/ µl when extraction performed using the lysis buffer BLM alone. Moreover, PCR amplification of 344 bp segment of the HBB showed a good quality of the extracted DNA. Conclusion: It was concluded that the superparamagneticbead based method is a reliable and effective method for DNA extraction from DBS and can be adopted for genetic diagnostic purposes.
Databáze: Directory of Open Access Journals
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