Single Lysis-Salting Out Method of Genomic DNA Extraction From Dried Blood Spots

Muntaj Shaik, Devaraju Kuramkote Shivanna, Mahesh Kamate, Vedamurthy Ab, Kruthika-Vinod Tp, Muntaj Shaik, Devaraju Kuramkote Shivanna, Mahesh Kamate, Vedamurthy Ab, Kruthika-Vinod Tp

Abstract

Background: Dried blood spots (DBS) are an important form of bio-sampling and valuable approach for storing blood samples for genetic studies. This has necessitated in developing an effective protocol to isolate genomic DNA (gDNA) from DBS samples.In this study, we have elucidated a dependable and non-hazardous "single lysis-salting out" (SLSO) protocol of gDNA extraction from DBS and compared against the available commercial kits.

Methods: For the purpose of this study, blood spots were collected on S&S 903 filter cards from 10 healthy volunteers and 30 patients with glutaric aciduria type I (GA-I). The gDNA was extracted from theseDBS samples by SLSO, QIAamp® gDNA Micro kit and innuPREP forensic kit methods. The quantity and quality of gDNA obtained from these methods were determined by measuring the absorbance using a Nanodrop spectrophotometer.

Results: The SLSO method showed four-fold and eight-fold increased yield of gDNA in healthy volunteers and patient samples, respectively, compared to commercial kits (p<0.0001). The protocol was also found to be cost efficient, reducing the per sample cost to almost half. The suitability of this method for genetic studies was confirmed by performing R402W genotyping by RFLP in GA-I patients. The genotyping results showed the presence of R402W mutation in 20% (6/30) of patients.

Conclusion: The SLSO method was found to be inexpensive, non-hazardous and a suitable technique for isolating gDNA from DBS samples for genetic studies.

Keywords: DNA isolation; GCDH; R402W mutation; genotyping; guthrie cards.

© 2016 Wiley Periodicals, Inc.

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Source: PubMed

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