Superparamagnetic-bead Based Method: An Effective DNA Extraction from Dried Blood Spots (DBS) for Diagnostic PCR
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 DN...
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| Format: | Article |
| Language: | English |
| Published: |
JCDR Research and Publications Private Limited
2014-04-01
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| Series: | Journal of Clinical and Diagnostic Research |
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| Online Access: | https://jcdr.net/articles/PDF/4226/8171_CE(Ra)_F(P)_PF1(PN)_PFA(P)_PF2(PAK)_PF2(PN).pdf |
| Summary: | 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. |
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| ISSN: | 2249-782X 0973-709X |