Muntaj Shaik1, Devaraju Kuramkote Shivanna2, Mahesh Kamate3, Vedamurthy Ab4, Kruthika-Vinod Tp5. 1. Department of Neurochemistry, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India. 2. Department of Biochemistry, Karnatak University Dharwad, Dharwad, Karnataka, India. 3. Department of Pediatrics, Jawaharlal Nehru Medical College, KLE University, Belgaum, Karnataka, India. 4. Department of Biotechnology & Microbiology, Karnatak University Dharwad, Dharwad, Karnataka, India. 5. Department of Neurochemistry, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India. kruthikav-tp@nimhans.ac.in, kruthikavinod@gmail.com.
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.
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.
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