There is no consensus on how to perform the manual extraction of nucleic acids from dried blood spots (DBSs). Current methods typically involve agitation of the DBSs in a solution for varying amounts of time with or without heat, and then purification of the eluted nucleic acids with a purification protocol. We explored several characteristics of genomic DNA (gDNA) DBS extraction such as extraction efficiency, the role of red blood cells (RBCs) in extraction and critical kinetic factors to understand if these protocols can be simplified while maintaining sufficient gDNA recovery. We found that agitation in a RBC lysis buffer before performing a DBS gDNA extraction protocol increases yield 1.5 to 5-fold, depending upon the anticoagulant used. The use of an alkaline lysing agent along with either heat or agitation was sufficient to elute quantitative polymerase chain reaction (qPCR) amplifiable gDNA in 5 minutes. This work adds insight into the extraction of gDNA from DBSs with the intention of informing a simple, standardized manual protocol for extraction.
Get full access to this article
View all access options for this article.
References
1.
ChoiEH, LeeSK, IhmC, et al.Rapid DNA extraction from dried blood spots on filter paper: Potential applications in biobanking. Osong Public Heal Res Perspect, 2014; 5(6):351–357.
2.
LimMD. Dried blood spots for global health diagnostics and surveillance: Opportunities and challenges. Am Soc Trop Med Hyg, 2018; 99(2):256–265.
3.
BjörkestenJ, EnrothS, ShenQ, et al.Stability of proteins in dried blood spot biobanks. Mol Cell Proteomics, 2017; 16(7):1286–1296.
Bou-ZeidW, BrutinD. Effect of relative humidity on the spreading dynamics of sessile drops of blood. Colloids Surfaces A Physicochem Eng Asp, 2014; 456(1):273–285; doi: 10.1016/j.colsurfa.2014.05.004
6.
CarpentieriD, ColvardA, PetersenJ, et al.Mind the quality gap when banking on dry blood spots. Biopreserv Biobank, 2021; 19(2):136–142.
7.
LoriaF, ManfrediM, Reverter-BranchatG, et al.Automation of RNA-based biomarker extraction from dried blood spots for the detection of blood doping. Bioanalysis, 2020; 12(11):729–736; doi: 10.4155/bio-2020-0041
8.
TuaillonE, KaniaD, PisoniA, et al.Dried blood spot tests for the diagnosis and therapeutic monitoring of HIV and viral hepatitis B and C. Front Microbiol, 2020; 11:373.
9.
LangPO, GovindS, DraméM, et al.Comparison of manual and automated DNA purification for measuring TREC in dried blood spot (DBS) samples with qPCR. J Immunol Methods, 2012; 384(1–2):118–127.
10.
NsojoA, AboudS, LyamuyaE. Comparative evaluation of amplicor HIV-1 DNA test, version 1.5, by manual and automated DNA extraction methods using venous blood and dried blood spots for HIV-1 DNA PCR testing. Tanzan J Health Res, 2010; 12(4):1–8.
11.
ScottLE, CrumpJA, MsuyaE, et al.Abbott RealTime HIV-1 m2000rt viral load testing: Manual extraction versus the automated m2000sp extraction. J Virol Methods, 2011; 172(1–2):78–80.
12.
TangN, PahalawattaV, FrankA, et al.HIV-1 viral load measurement in venous blood and fingerprick blood using Abbott RealTime HIV-1 DBS assay. J Clin Virol, 2017; 92:56–61; doi: 10.1016/j.jcv.2017.05.002
13.
PandaBB, MeherAS, HazraRK. Comparison between different methods of DNA isolation from dried blood spots for determination of malaria to determine specificity and cost effectiveness. J Parasit Dis, 2019; 43(3):337–342; doi: 10.1007/s12639-019-01136-0
14.
HueNT, PhongPT, DieuN, et al.An efficiency human genomic DNA extraction from dried blood spots. Proc Environ Sci, 2011; 8:179–185; doi: 10.1016/j.proenv.2011.10.029
15.
PoulsenJB, LescaiF, GroveJ, et al.High-quality exome sequencing of whole-genome amplified neonatal dried blood spot DNA. PLoS One, 2016; 11(4):e0153253.
16.
RajatilekaS, LuytK, El-BokleM, et al.Isolation of human genomic DNA for genetic analysis from premature neonates: A comparison between newborn dried blood spots, whole blood and umbilical cord tissue. BMC Genet, 2013; 14:105.
17.
SimonN, ShallatJ, Williams WietzikoskiC, et al.Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots. Biol Methods Protoc, 2021; 5(1):1–7.
18.
WijnenPAHM, Op den BuijschRAM, CheungSCY, et al.Genotyping with a dried blood spot method: A useful technique for application in pharmacogenetics. Clin Chim Acta, 2008; 388(1–2):189–191.
19.
St. Julien KR, Jelliffe-Pawlowski LL, Shaw GM, et al. High quality genome-wide genotyping from archived dried blood spots without DNA amplification. PLoS One, 2013; 8(5):e64710; doi: 10.1371/journal.pone.0064710
Ait BelkacemI, Mossadegh-KellerN, BourgoinP, et al.Cell analysis from dried blood spots: New opportunities in immunology, hematology, and infectious diseases. Adv Sci, 2021; 8(18):1–10.
22.
SharmaA, JaiswalS. Dried blood spots: Concepts, present status, and future perspectives in bioanalysis. Drug Test Anal, 2014; 6(5):399–414.
23.
AgrawalP, KatragaddaS, HariharanAK, et al.Validation of whole genome sequencing from dried blood spots. BMC Med Genomics, 2021; 14(1):110; doi: 10.1186/s12920-021-00951-w
24.
BassaganyasL, FreedmanG, VakaD, et al.Whole exome and whole genome sequencing with dried blood spot DNA without whole genome amplification. Hum Mutat, 2018; 39(1):167–171; doi: 10.1002/humu.23356
25.
GrünerN, StambouliO, RossRS. Dried blood spots—Preparing and processing for use in immunoassays and in molecular techniques. J Vis Exp, 2015; 2015(97):52619.
26.
MössnerBK, StaugaardB, JensenJ, et al.Dried blood spots, valid screening for viral hepatitis and human immunodeficiency virus in real-life. World J Gastroenterol, 2016; 22(33):7604.
27.
BucktonAJ, BissettSL, MyersRE, et al.Development and optimization of an internally controlled dried blood spot assay for surveillance of human immunodeficiency virus type-1 drug resistance. J Antimicrob Chemother, 2008; 62(6):1191–1198; doi: 10.1093/jac/dkn412
KantorR, DeLongA, BalamaneM, et al.HIV diversity and drug resistance from plasma and non-plasma analytes in a large treatment programme in western Kenya. J Int AIDS Soc, 2014; 17(1):1–12.
30.
GuichetE, SerranoL, LaurentC, et al.Comparison of different nucleic acid preparation methods to improve specific HIV-1 RNA isolation for viral load testing on dried blood spots. J Virol Methods, 2018; 251:75–79.
31.
SoetensO, Vauloup-FellousC, FoulonI, et al.Evaluation of different cytomegalovirus (CMV) DNA PCR protocols for analysis of dried blood spots from consecutive cases of neonates with congenital CMV infections. J Clin Microbiol, 2008; 46(3):943–946.
32.
FelicielloI, ChinaliG. A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli. Anal Bioanal Chem, 1993; 212:394–401.
33.
Saavedra-MatizCA, IsabelleJT, BiskiCK, et al.Cost-effective and scalable DNA extraction method from dried blood spots. Clin Chem, 2013; 59(7):1045–1051.
34.
LeeK, MurphyJ, TripathiA. Electro-DBS: A simple method to rapidly extract genomic DNA from dried blood spots. Anal Chem, 2022; 94(39):13404–13412; doi: 10.1021/acs.analchem.2c02021
35.
MachadoMC, VimbelaGV, NilssonM, et al.Rapid electrophoretic recovery of DNA from dried blood spots. Electrophoresis, 2019; 40(14):1812–1819.
36.
DriverGA, PattonJC, MoloiJ, et al.Low risk of contamination with automated and manual excision of dried blood spots for HIV DNA PCR testing in the routine laboratory. J Virol Methods, 2007; 146(1–2):397–400.
37.
LeeK, TripathiA. Parallel DNA extraction from whole blood for rapid sample generation in genetic epidemiological studies. Front Genet, 2020; 11(374):1–16; doi: 10.3389/fgene.2020.00374/full
38.
Al-soudWA, RadstromP. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol, 2001; 39(2):485–493.
39.
ChalmersC, RussellWJ. When does blood haemolyse?: A temperature study. Br J Anaesth, 1974; 46(10):742–746; doi: 10.1093/bja/46.10.742
40.
OellingrathIM. Heat degradation of heme in methemoglobin and metmyoglobin model systems measured by reversed-phase ion-pair high performance liquid chromatography. J Food Sci, 1988; 53(1):40–42; doi: 10.1111/j.1365-2621.1988.tb10173.x
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
