Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

Abstract Dried blood spots (DBS) are widely utilized as part of universal newborn screening and as a means of transporting samples from field sites. We use DBS from African field sites to assess for rare maternal-fetal cell exchange during pregnancy known as microchimerism. We aimed to develop a protocol to maximize the quantity of high-quality genomic DNA (gDNA) extracted from DBS. The total gDNA yield obtained from control DBS utilizing a Qiagen-based protocol and a Chelex® 100 resin-based protocol was first compared. Variations of the Chelex® protocol were subsequently tested to develop an optimized protocol. The gDNA was quantified by qPCR targeting the human beta-globin gene. DNA yield for a given experimental condition was normalized to a Chelex® control performed on the same day, and the total yields were compared using a Student’s t-test. The control Chelex® protocol yielded 590% more DNA than the QIAamp® DNA Blood Mini Kit . The absolute efficiency of the control Chelex® protocol was 54%, compared to an absolute efficiency of 9% for the QIAamp® DNA Blood Mini Kit. Modification of the Chelex® protocol to include a second heat precipitation from the same DBS increased the gDNA yield by 29% (P < 0.001). Our optimized protocol including this modification increased the absolute efficiency of extraction to 68%. The gDNA extracted using the Chelex® protocol was stable through repeated freeze–thaw cycles. In a mock microchimerism experiment, rare donor alleles at a frequency of 10 in 100 000 could be identified in gDNA from DBS extracted using the optimized Chelex® protocol. Our findings may be of significance for a diverse range of applications that utilize DBS and require high-quality DNA, including newborn screening programs, pathogen and drug resistance screening from remote field sites, forensics, and rare allele detection.

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Measurement of psychosine in dried blood spots — a possible improvement to newborn screening programs for Krabbe disease

Journal of Inherited Metabolic Disease ◽

10.1007/s10545-015-9822-z ◽

2015 ◽

Vol 38 (5) ◽

pp. 923-929 ◽

Cited By ~ 46

Author(s):

Coleman T. Turgeon ◽

Joseph J. Orsini ◽

Karen A. Sanders ◽

Mark J. Magera ◽

Thomas J. Langan ◽

...

Keyword(s):

Newborn Screening ◽

Dried Blood Spots ◽

Krabbe Disease ◽

Screening Programs ◽

Blood Spots

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Storage and use of residual dried blood spots from state newborn screening programs

The Journal of Pediatrics ◽

10.1016/j.jpeds.2005.12.053 ◽

2006 ◽

Vol 148 (5) ◽

pp. 618-622 ◽

Cited By ~ 49

Author(s):

Richard S. Olney ◽

Cynthia A. Moore ◽

Jelili A. Ojodu ◽

Mary Lou Lindegren ◽

W. Harry Hannon

Keyword(s):

Newborn Screening ◽

Dried Blood Spots ◽

Screening Programs ◽

Blood Spots

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Next-generation sequencing as a second-tier diagnostic test for newborn screening

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2018-0088 ◽

2018 ◽

Vol 31 (8) ◽

pp. 927-931 ◽

Cited By ~ 2

Author(s):

Xiaomei Luo ◽

Ruifang Wang ◽

Yanjie Fan ◽

Xuefan Gu ◽

Yongguo Yu

Keyword(s):

Next Generation Sequencing ◽

Newborn Screening ◽

Diagnostic Test ◽

Genomic Dna ◽

Sanger Sequencing ◽

Dried Blood Spots ◽

Next Generation ◽

Blood Spots ◽

Second Tier ◽

Generation Sequencing

Abstract Background Tandem mass spectrometry (MS/MS) has been used for newborn screening (NBS) of inherited metabolic diseases (IMDs) for decades. However, the traditional approach can yield false-positive or false-negative results and is affected by biochemical substrate-level fluctuations. To overcome the current limitations, we explored the possibility of using next-generation sequencing (NGS) as a second-tier diagnostic test to detect gene mutations in samples with abnormal MS/MS results. Methods Genomic DNA was extracted from dried blood spots and we designed a multigene panel, comprising 77 genes related to over 40 IMDs, for NBS. The prepared libraries were sequenced on the Ion Personal Genome Machine (PGM) platform. Thirty-eight samples identified as abnormal by MS/MS were tested for the diagnostic accuracy of NGS compared with Sanger sequencing. Results The concentration of DNA extracted from the 38 dried blood spots was sufficient for library preparation. The coverage and depth of the sequencing data were sufficient for the analysis. For all samples, the NGS results were consistent with the Sanger sequencing results. Conclusions The genomic DNA extracted from dried blood spots could be used for NGS, generating reliable sequencing results, and NGS may function as a second-tier diagnostic test for NBS. Ion PGM could facilitate the molecular diagnosis of IMDs with appropriate primers designed for candidate genes.

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Determination of Acid α-Glucosidase Activity in Blood Spots as a Diagnostic Test for Pompe Disease

Clinical Chemistry ◽

10.1093/clinchem/47.8.1378 ◽

2001 ◽

Vol 47 (8) ◽

pp. 1378-1383 ◽

Cited By ~ 61

Author(s):

Kandiah Umapathysivam ◽

John J Hopwood ◽

Peter J Meikle

Keyword(s):

Newborn Screening ◽

Pompe Disease ◽

Dried Blood Spots ◽

Cultured Fibroblasts ◽

Virtual Absence ◽

Screening Programs ◽

Blood Spots ◽

Enzyme Replacement ◽

Glucosidase Activity

Abstract Background: Pompe disease is an autosomal recessive disorder of glycogen metabolism that is characterized by a deficiency of the lysosomal acid α-glucosidase. Enzyme replacement therapy for the infantile and juvenile forms of Pompe disease currently is undergoing clinical trials. Early diagnosis before the onset of irreversible pathology is thought to be critical for maximum efficacy of current and proposed therapies. In the absence of a family history, the presymptomatic detection of these disorders ideally can be achieved through a newborn-screening program. Currently, the clinical diagnosis of Pompe disease is confirmed by the virtual absence, in infantile onset, or a marked reduction, in juvenile and adult onset, of acid α-glucosidase activity in muscle biopsies and cultured fibroblasts. These assays are invasive and not suited to large-scale screening. Methods: A sensitive immune-capture enzyme activity assay for the measurement of acid α-glucosidase protein was developed and used to determine the activity of this enzyme in dried-blood spots from newborn and adult controls, Pompe-affected individuals, and obligate heterozygotes. Results: Pompe-affected individuals showed an almost total absence of acid α-glucosidase activity in blood spots. The assay showed a sensitivity and specificity of 100% for the identification of Pompe-affected individuals. Conclusions: The determination of acid α-glucosidase activity in dried-blood spots is a useful, noninvasive diagnostic assay for the identification of Pompe disease. With further validation, this procedure could be adapted for use with blood spots collected in newborn-screening programs.

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DNA from dried blood spots yields high quality sequences for exome analysis

10.1101/2020.05.19.105304 ◽

2020 ◽

Cited By ~ 1

Author(s):

Uma Sunderam ◽

Aashish N. Adhikari ◽

Kunal Kundu ◽

Jennifer M. Puck ◽

Robert Currier ◽

...

Keyword(s):

Dna Damage ◽

Reference Genome ◽

Variant Calling ◽

Dried Blood Spots ◽

Sequencing Data ◽

High Quality ◽

Potential Health ◽

Screening Programs ◽

Blood Spots ◽

Variant Discovery

AbstractBackgroundDNA sequencing of archived dried blood spots (DBS) collected by newborn screening programs constitutes a potential health resource to study newborn disorders and understand genotype-phenotype relationships. However, its essential to verify that sequencing reads from DBS derived DNA are suitable for variant discovery.ResultsWe explored 16 metrics to comprehensively assess the quality of sequencing reads from 180 DBS and 35 whole blood (WB) samples. These metrics were used to assess a) mapping of reads to the reference genome, b) degree of DNA damage, and c) variant calling. Reads from both sets mapped with similar efficiencies, had similar overall DNA damage rates, measured by the mismatch rate with the reference genome, and produced variant calls sets with similar Transition-Transversion ratios. While evaluating single nucleotide changes that may have arisen from DNA damage, we observed that the A>T and T>A changes were more frequent in DNA from DBS than from WB. However, this did not affect the accuracy of variant calling, with DBS samples yielding a comparable count of high quality SNVs and indels in samples with at least 50x coverage.ConclusionsOverall, DBS DNA provided exome sequencing data of sufficient quality for clinical interpretation.

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Storing Newborn Blood Spots: Modern Controversies

The Journal of Law Medicine & Ethics ◽

10.1111/j.1748-720x.2004.tb01979.x ◽

2004 ◽

Vol 32 (4) ◽

pp. 741-748 ◽

Cited By ~ 15

Author(s):

Linda Kharaboyan ◽

Denise Avard ◽

Bartha Maria Knoppers

Keyword(s):

Newborn Screening ◽

Diagnostic Test ◽

Clinical Data ◽

Dried Blood Spots ◽

Parental Consent ◽

Public Attention ◽

Screening Programs ◽

Blood Spots ◽

Blood Spot

Though in existence for over thirty-five years, due to the increasing panoply of possible tests. Newborn screening programs are drawing public attention. Many jurisdictions have mandatory newborn screening programs for treatable disorders. Disorders are detected through tests on blood spots drawn from a newborn’s heel soon after birth and verified through a diagnostic test with follow-up. Unbeknownst to most parents, these blood spot cards are also stored thereafter. Indeed, while dried blood spots (DBSs) are primarily used for screening for health problems, experience demonstrates that they can be made useful in various contexts unrelated to screening.Newborn dried blood spots have taken on a new life as a result of developments in genetics and the increasing ability of bioinformatics to link DNA information with clinical data. Additionally, storage and secondary uses have been documented to occur without parental consent.

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A Comparative Analysis of the Governance and Use of Residual Dried Blood Spots from State Newborn Screening Programs and Neonatal Biobanks

Journal of Empirical Research on Human Research Ethics ◽

10.1525/jer.2013.8.3.22 ◽

2013 ◽

Vol 8 (3) ◽

pp. 22-33 ◽

Cited By ~ 1

Author(s):

Elicia D. Preslan ◽

Debra J. H. Mathews

Keyword(s):

Comparative Analysis ◽

Newborn Screening ◽

Dried Blood Spots ◽

Screening Programs ◽

Blood Spots

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Comparison between Enzyme Immunoassays Performed on Samples of Dried Blood and Serum for Toxoplasmosis Prenatal Screening: Population-based Study

Revista Brasileira de Ginecologia e Obstetrícia / RBGO Gynecology and Obstetrics ◽

10.1055/s-0041-1730285 ◽

2021 ◽

Vol 43 (05) ◽

pp. 351-356

Author(s):

Bárbara Araújo Marques ◽

Ericka Vianna Machado Carellos ◽

Vânia Maria Novato Silva ◽

Fernando Henrique Pereira ◽

Maria Regina Lage Guerra ◽

...

Keyword(s):

Pregnant Women ◽

Prenatal Screening ◽

Dried Blood Spots ◽

Population Based ◽

Minas Gerais ◽

Immunoglobulin M ◽

Screening Tests ◽

Reference Test ◽

Screening Programs ◽

Blood Spots

Abstract Objective Most prenatal screening programs for toxoplasmosis use immunoassays in serum samples of pregnant women. Few studies assess the accuracy of screening tests in dried blood spots, which are of easy collection, storage, and transportation. The goals of the present study are to determine the performance and evaluate the agreement between an immunoassay of dried blood spots and a reference test in the serum of pregnant women from a population-based prenatal screening program for toxoplasmosis in Brazil. Methods A cross-sectional study was performed to compare the immunoassays Imunoscreen Toxoplasmose IgM and Imunoscreen Toxoplasmose IgG (Mbiolog Diagnósticos, Ltda., Contagem, Minas Gerais, Brazil)in dried blood spots with the enzyme-linked fluorescent assay (ELFA, BioMérieux S.A., Lyon, France) reference standard in the serum of pregnant women from Minas Gerais Congenital Toxoplasmosis Control Program. Results The dried blood spot test was able to discriminate positive and negative results of pregnant women when compared with the reference test, with an accuracy of 98.2% for immunoglobulin G (IgG), and of 95.8% for immunoglobulin M (IgM). Conclusion Dried blood samples are easy to collect, store, and transport, and they have a good performance, making this a promising method for prenatal toxoplasmosis screening programs in countries with continental dimensions, limited resources, and a high prevalence of toxoplasmosis, as is the case of Brazil.

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