A simple method for extraction of DNA from Guthrie cards.

Schneeberger, Christian; Kury, F; Larsen, Jörg; Speiser, Paul; Zeillinger, Robert

doi:10.1101/gr.2.2.177

Cited by 12 publications

(9 citation statements)

References 5 publications

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“…This observation may result from better preservation and/or increased yield of template DNA in the presence of GT (11,12,24), GT-mediated inhibition of endogenous nucleases (12, 13), or some combination thereof. Although comparable amounts of Std-903 and GT-903 were used for amplification, it was impossible to quantitate the DNA that was tightly bound to Std-903.…”

Section: Discussionmentioning

confidence: 94%

“…The role of guanidine thiocyanate in the differential binding of DNA and PCR inhibitors to the filter paper matrix is unclear. Guanidine thiocyanate has been a widely used chaotrope for DNA and RNA purification (11,12,24). Although GT has been reported to enhance the affinity of DNA for silica and glass particles (11,12), it appears to prevent or at least limit DNA interaction with the filter paper matrix while promoting inhibitor binding.…”

Section: Discussionmentioning

confidence: 99%

“…Guanidine thiocyanate has been a widely used chaotrope for DNA and RNA purification (11,12,24). Although GT has been reported to enhance the affinity of DNA for silica and glass particles (11,12), it appears to prevent or at least limit DNA interaction with the filter paper matrix while promoting inhibitor binding. The lack of elution of both high (protein) and low molecular (iron, heme) components from GT-903 would appear to suggest that these components have become irreversibly bound or fixed in the presence of guanidine thiocyanate.…”

Section: Discussionmentioning

confidence: 99%

“…Amplification of Guthrie card DNA presents considerable technical problems due to the presence of high levels of natural whole blood PCR inhibitors (heavy metals, protein, heme, and heme degradation products) and low copy number of template (5)(6)(7)(8)(9)(10). To overcome these limitations, investigators have resorted to a variety of DNA extraction methods using organic solvents (phenol/chloroform/isoamyl alcohol) (4, 10, chaotropic agents (cesium trifluoroacetate, guanidine-thiocyanate and -HCl) (11)(12)(13) and detergents (sodium dodecyl sulfate) (3,4,10). Unfortunately, these harsh extraction methods generally result in the simultaneous release of nucleic acid and PCR inhibitors.…”

Section: Introductionmentioning

confidence: 99%

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Amplification of Guthrie card DNA: Effect of guanidine thiocyanate on binding of natural whole blood PCR inhibitors

Makowski

Davis

Hopfer

1997

J. Clin. Lab. Anal.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Amplification of Guthrie card DNA: Effect of guanidine thiocyanate on binding of natural whole blood PCR inhibitors

Makowski

Davis

Hopfer

1997

J. Clin. Lab. Anal.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Sirdah¹

2014

JCDR

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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...

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Human DNA Sampling and Banking

Spanakis

2002

Molecular Genetic Epidemiology — A Laboratory Perspective

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A simple method for extraction of DNA from Guthrie cards.

Cited by 12 publications

References 5 publications

Amplification of Guthrie card DNA: Effect of guanidine thiocyanate on binding of natural whole blood PCR inhibitors

Amplification of Guthrie card DNA: Effect of guanidine thiocyanate on binding of natural whole blood PCR inhibitors

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

Human DNA Sampling and Banking

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