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

Abstract

IntrOductIOnDried blood samples spotted on filter papers, introduced by Robert Guthrie in 1963, have been widely used in neonatal screening programs for the early identification of congenital disorders and presymptomatic management of affected neonates [1][2][3]. The DBS were first used in the early 1970s in the neonatal screening for phenylketonuria (PKU) which provided a simple, inexpensive and unique method for heel-stick blood collection from neonates onto a special cotton fiber filter-paper which is still known as a "PKU card" or "Guthrie card" [4,5]. Since then, DBS have been collected routinely from babies not only for PKU screening but also for other biochemical screenings associated with congenital, inherited, and infectious disorders [6][7][8]. The success of these DBS as an easy and cost-effective method for collecting, archiving, and transporting blood specimens has inspired researchers to explore better DNA extraction and PCR-based molecular testing methods for these samples [9][10][11][12]. The DBS sampling is an effective alternative for lymphocyte pellets because DBS sampling requires only a few droplets of whole blood, not necessarily venous blood, and can be directly spotted on Guthrie cards or filter papers [13][14][15].The commonly used methods and techniques for extracting genomic DNA from DBS are varied in terms of extraction principle, reagents used, and quantity and quality of the extracted DNA. Manual protocols include the extraction of DNA with Tris-EDTA buffer, methanol, and Chelex-100 [16][17][18][19]. In addition, commercially available kits for DNA extraction from DBS are available [11,20], but their cost could limit their use for large scale-screening programs.Paramagnetic-bead based extraction method of DNA from body fluids such as semen, blood, vaginal, and buccal cells has been established and resulted in a cost-efficient and high-throughput DNA extraction method that can be used for PCR-based molecular Pathology Section testing [21][22][23][24]. The present work explores and evaluates the effectiveness of a superparamagnetic-bead based method (LGC Genomic, Germany) in extracting DNA from DBS. MAterIAls And MethOdsWhole blood samples collected from 16 apparently healthy subjects and two ß-thalassemia patients were used for spotting onto filter papers, direct DNA extraction and quantitative and qualitative evaluations. spotting onto filter papersFrom each sample four 100 µl applications of whole blood were spotted onto Ahlstrom 226 grad newborn screening filter papers (ID Biological Systems, Greenville, SC, USA), and were dried at ambient temperature (28-30 0 C) for one week. dnA extractionDNA extraction from whole blood and DBS were performed using the superparamagnetic-bead based DNA extraction kit manufactured by LGC Genomic, formerly AGOWA, Germany. The extraction was carried out according to the manufacturer's instruction for whole blood and with some modifications for extraction from DBS. The standard superparamagnetic-bead based DNA extraction protocol includes four princi...

Rapid screening of UPB1 gene variations by high resolution melting curve analysis

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Optimization of extraction of genomic DNA from archived dried blood spot (DBS): potential application in epidemiological research & bio banking

“…Several papers have been published on extraction of DNA from DBS, some authors have described increasing the yield by employing various means 1 - 2 - 3 . A mention of these papers in the discussion would help the readers.…”