reviewarticle - oswaldo cruz foundation

Review ArticleCurrent Nucleic Acid Extraction Methods andTheir Implications to Point-of-Care Diagnostics

Nasir Ali12 Rita de Caacutessia Pontello Rampazzo2

Alexandre Dias Tavares Costa23 andMarco Aurelio Krieger123

1Departamento de Engenharia de Bioprocessos e Biotecnologia Universidade Federal do Parana (UFPR) Curitiba PR Brazil2Instituto de Biologia Molecular do Parana (IBMP) Fiocruz Curitiba PR Brazil3Instituto Carlos Chagas (ICC) Fiocruz Curitiba PR Brazil

Correspondence should be addressed to Alexandre Dias Tavares Costa alexandrecostafiocruzbr

Received 31 March 2017 Accepted 5 June 2017 Published 12 July 2017

Academic Editor Francesco Dondero

Copyright copy 2017 Nasir Ali et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for many downstream applications Manymodifications have been introduced to the original 1869 method Modern processes are categorized into chemical or mechanicaleachwith peculiarities that influence their use especially in point-of-care diagnostics (POC-Dx) POC-Dx is a new approach aimingto replace sophisticated analytical machinery with microanalytical systems able to be used near the patient at the point of careor point of need Although notable efforts have been made a simple and effective extraction method is still a major challengefor widespread use of POC-Dx In this review we dissected the working principle of each of the most common NAE methodsoverviewing their advantages and disadvantages as well their potential for integration in POC-Dx systems At present it seemsdifficult if not impossible to establish a procedure which can be universally applied to POC-Dx We also discuss the effects ofthe NAE chemicals upon the main plastic polymers used to mass produce POC-Dx systems We end our review discussing thelimitations and challenges that should guide the quest for an efficient extractionmethod that can be integrated in a POC-Dx system

1 Introduction

Nucleic acid extraction (NAE) is one of the most pivotalsteps in molecular biology being routinely used in manyareas of the biological andmedical sciences as this proceduremarks a starting point in anymolecular diagnostic kit [1]Thiscrucial procedure has been known for over a century and hasdeveloped substantially over the last decades However someprogress still has to be achieved so that NAE protocols leavethe laboratory settings into the ldquoreal worldrdquo of point-of-carediagnostics (POC-Dx)

Nowadays it is known that intracellular nucleic acids(NAs) may be broadly categorized as genomic (or chromo-somal) plasmids and different types of RNAs [2] AlthoughRNAs possess uracil while DNAs present thymine [3]nucleic acids exhibit similar basic biochemical properties butmight have quite distinct tridimensional structures (genomicplasmid tRNA mRNA rRNA etc) However despite the

structural differences the most commonly used methodsdescribed in the present text can be applied to DNA in itsmany organizational formats (chromosomal plasmid etc)as well as RNA and its multidimensional formats (mRNArRNA tRNAmiRNA etc) withminormodifications [1 4 5]

NAE can be roughly divided into four steps which canbe modulated depending on the sample and downstreamapplications (i) cell disruption (ii) removal of membranelipids proteins and other nucleic acids (iii) nucleic acidpurificationbinding from bulk and (iv) nucleic acid concen-tration [6]

Cell disruption or disintegration can be achieved byphysical andor chemical methods whose main aim is todisrupt the cell wall andor cellular membranes Disruptionmethods aremainly based on properties of the sample and forthis purpose a wide range of tools and approaches are usedeither alone or combined to achieve tissuecell disruption[7] Lytic enzymes chaotropic agents and different types

HindawiBioMed Research InternationalVolume 2017 Article ID 9306564 13 pageshttpsdoiorg10115520179306564

2 BioMed Research International

Table 1 Main characteristics of chemical and mechanical methods to extract nucleic acid (adapted from Harrison 2003)

Method Technique Principle Mode of lysis Cost Most usualapplication References

Chemical

Osmotic shock Osmotic rupture ofmembrane Gentle Cheap Spheroplasts and

Protoplasts [9]

Enzymaticdigestion

Digestion of cellwall Gentle

Cheap at smallscale expensive atlarge scale

Gram-positive andGram-negativebacteria

[13]

Detergents Solubilization ofmembranes Gentle Moderate General use [14]

Alkali treatment Solubilization ofmembrane Harsh Cheap Plasmid DNA [15]

Mechanical

Homogenization(blade or pestle) Shredding of cells Moderate

Moderate (methodof choice for largescale)

Animal tissues [10]

Ultrasonication orcavitation

Disruption of cellsby pressure Harsh Moderate to

expensive

Good forspheroplasts butnot primary cells

[11 12]

Pressure cell(ldquoFrench pressrdquo)

Disruption of cellsby shear force Harsh Moderate

Used forGram-negative andsomeGram-positivebacteria

[6]

Ball millCells crushedbetween glasssteelballsbeads

Harsh Cheap

Used for bacteriayeast microalgaeunicellular animalcells

[15]

of detergents are the main components of chemical lysiswhile mechanical method disrupts the cells by grindingshearing bead beating and shocking [8] It is interestingto note that if one technique does not yield good resultsanothermight prove successful Osmotic shockmethods haveyielded in certain cases better results than common NApurifications protocols such as phenol-chloroform extractionand bead beating [9] Not only is cell disruption importantfor DNA extraction but it also plays a crucial role in thebiopharmaceutical industry as many recombinant proteinsand other important constituents of the cell can be recoveredthrough this process [10ndash12] Another approach for celldisruption is the use of different methods in combinationA good example is the case for enzymatic lysis where manyprotocols use proteases to free the NA from its protectiveprotein scaffold Also the inactivation of cellular nucleasesthat come free into solution in order to protect the newprotein-free NA is crucial [13] A combination of detergentsand chaotropic salts in a single solution is used to solubilizecell wall and or cell membrane and inactivate intracellularnucleases [14 15] Mechanical disruption on the other handmakes use of force to extract out constituents of the cell Aclassic example of grinding in biosciences is the use of mortarand pestle [6] which is nowadays optimized with the useof liquid nitrogen (when allowed by the sample) Cells wallscan also be disrupted by the shock waves created by rapidchanges in pressure elicited by sonication or cavitation [16ndash18] Other mechanical tools available for cell disruption areshearing which use a tangential force to make a hole in the

cell [6] and bead beating which uses different glass or steelbeads to rupture tough cell wall as mentioned by Bunge et al[19] These processes are briefly summarized in Table 1 withconsolidated examples

NAE methods encompass extraction of both DNA andRNA but can be more broadly characterized into chemicallydriven or solid-phase methods both contain the four stepsmentioned above [1 4 5] In the next sections we willreview the working principle of andor rationale for the mainmethods used nowadays in the biological and medical sci-ences Since molecular diagnostics rely heavily on techniquesthat start with NAE we will also discuss some of the basicfeatures of devices available for POC-Dx culminating withthe challenges and limitations of adapting NAE methods topoint-of-care diagnostic tests

2 Chemically Driven Methods

These methods rely on biochemical properties of the cellularcomponents to elicit the desired molecular separation andmight exhibit preference or exclusivity in extracting DNA orRNA depending on its intrinsic characteristics

21 Cesium Chloride (CsCl) Gradient Centrifugation withEthidium Bromide (EtBr) This technique is mainly based onthe phenomenon of buoyant and specific density Ethidiumbromide (EtBr) is an intercalating agent thus reportingthe location of the double-stranded DNA under UV-lightand allowing the easy visual separation of the supercoiled

BioMed Research International 3

and nonsupercoiled DNA molecules The basic mechanismby which EtBr separates the two molecules is decreasingthe buoyant density of comparatively linear molecules [20]After the ultracentrifugation CsCl has to be dialyzed of thecollectedDNAThemethod can be used to extract DNA frombacteria although a large-scale culture is needed [21] thismethod can be used for purification of various forms ofDNAsuch as chromosomal plasmid DNA rDNA or mitochon-drial DNA [22] Being sensitive and provider of good yieldsof pure DNA the method is laborious time-consuming andcostly as compared to other purification protocols Further-more EtBR can affect downstream applications such as PCRcloning and DNA sequencing [23] There is concern aboutusing EtBr which is known to cause genotoxicity and frameshift mutations For mice nontoxic doses up to 50mgkghave been used for cattle up to 1mgkg of body weightHowever the concentration used in gel staining solutions(025ndash1 120583gmL) is below the level of toxicity even though careis suggested in handling EtBr [23 24]

22GuanidiniumThiocyanate-Phenol-ChloroformExtractionA guanidinium thiocyanate- (GuSCN-) phenol-chloroformmixture allows for RNA extraction in a single-step procedureas demonstrated by Chomczynski and Sacchi [25] Prior tothe development of guanidinium method phenol extractionwas normally used for extraction in a two-step laboriousprocess The method was modified successively over timestarting from Ullrich et al [26] who used guanidinium thio-cyanate instead of guanidinium chloride for RNA isolationfollowed later on by Chirgwin et al in 1979 [27] usingGuSCNcombined with extended hours of ultracentrifugation anda CsCl cushion In order to enhance the quality of thefinal nucleic acid the technique was improved by usingguanidinium thiocyanate and phenol-chloroform with ashorter centrifugation time [28] Despite being less solublein water than guanidine hydrochloride another commonsalt of guanidine GuSCN has stronger denaturing propertiesbecause both its ions are chaotropic

The basic principle of the method is the separation ofRNA from DNA and proteins after extraction with an acidicsolution which consists mainly of GuSCN sodium acetatephenol and chloroform followed by centrifugation TotalRNA remains in the upper aqueous phase while most ofDNA and proteins part remain either in the interphase or inthe lower organic phase under acidic condition Total RNAis then recovered through precipitation by isopropanol andcan be used for subsequent process The original methodwas carried out in mammalian tissue but later on it hasbeen used for plants with some modification [29] animals[27] and cultured cell tissues as well [28 30] OptimumpH plays a critical role in the separation process as DNApartitions to the organic phase under acidic condition (pH4ndash6) or to the aqueous phase at neutral pH (pH7-8)Themaindrawback of this method is that phenol and chloroform areboth hazardous chemicals [28] This reagent is commerciallyavailable with different names such as Sigma-Aldrich TRIReagent and Thermo Fisher TRIzol Reagent High purityand yield of the extracted NA are the hallmark of thisprocedure

23 Cetyltrimethylammonium Bromide (CTAB) ExtractionCetyltrimethylammonium bromide extraction method ismainly used for plant samples and their parts such as leavesseeds and grains The method is used for various foodsamples as well The basic composition of CTAB extractionbuffer includes 2 CTAB at alkaline pH but like many otherextraction protocols CTAB has been modified according tothe need of each sample [31] CTAB works by precipitatingnucleic acids and acidic polysaccharides in low ionic strengthsolutions while proteins and neutral polysaccharides remainin solution Next the CTAB-nucleic acid precipitated com-plex is solubilized at high-salt concentrations leaving theacid polysaccharides in the precipitate [1] During the pre-cipitation and washing steps CTAB method uses variousorganic solvents and alcohols such as phenol chloroformisoamyl alcohol and mercaptoethanol The main drawbackof this procedure is that it is time-consuming and makes useof toxic chemicals like phenol and chloroform MoreoverCTAB extracted DNA requires further purification to avoidinhibition of PCR analyzes [32]

24 Chelex Extraction Chelex (Bio-Rad Laboratories CAUSA) is a chelating resin frequently used in the field offorensics for DNA extraction from various sources such ashair blood stain cards and buccal swabs [33] Accordingto [33] boiling in the presence of Chelex can increasethe signal during PCR amplification of relatively minoramount of DNA possibly by inhibiting DNA degradation bychelating metal ions which cause DNA breakdown at hightemperature and lower ionic conditions Chelex is a styrenedivinylbenzene copolymer containing paired iminodiacetateions which are used as chelators for polyvalent metal ions[34] This technique is interesting as it is quick has fewmanipulating steps and does not use hazardous chemicalssuch as phenolchloroform Its main drawback is the inabilityto efficiently remove PCR inhibitors from complex samplesdue to the lack of purification steps [35] This method is alsonot suitable for restriction fragment length polymorphism(RFLP) analyses because exposure of DNA to the high tem-perature and alkalinity of this protocol results in denaturationand breakage of DNA

25 Alkaline Extraction Alkaline extraction method is ded-icated to plasmid DNA isolation described by Bimboim andDoly [36] The basic principle of this method is selectivealkaline denaturation of highmolecular weight chromosomalDNA while covalently bond circular plasmid DNA remainsintact After neutralization chromosomal DNA renaturesand makes an insoluble precipitate while plasmid DNAremains in the supernatant This method is useful for bothsmall and large DNA plasmids [36]

The method involves harvesting the bacteria of interestfrom culture media and exposing them to alkaline solu-tion (consisting basically of SDS and NaOH) SDS act asdetergent to lyse the cells and denature proteins whilealkaline condition denatures genomic DNA plasmid DNAand proteins Potassium acetate (pH 52) addition neutral-izes the mixture and results in renaturation of plasmidas well as genomic DNA Further addition of ethanol (or


Table 2 Summary of advantages and disadvantages of the main NAEmethods GuSCN guanidine thiocyanate CsCl cesium chloride EtBrethidium bromide CTAB cetyltrimethylammonium bromide

Method Advantage Disadvantage Reference(1) GuSCN-phenol- chloroformextraction

High purity and yield of DNA orRNA Hazardous chemicals [21 23]

(2) Alkaline extraction Fastest reliable and relatively easyprocedure

Medium purity and fragmentation ofgenomic DNA [26]

(3) CsCl gradient centrifugation withEtBr

High purity and yield of DNA orRNA

Laborious costly and timeconsuming [29 30]

(4) Oligo(dT) cellulosechromatography

Fast protocol good yield of mRNArecovery Purification bias for mRNAs [1]

(5) Chelex extraction Quick and simple protocol no use ofhazardous chemicals Low purity of nucleic acids [35 36]

(6) CTAB extraction Efficient method for plant and otherldquohard to lyserdquo samples

Laborious time-consuming use ofhazardous chemicals [38]

isopropanol) precipitates genomic DNA while plasmid DNAcan be collected from the supernatant after a short 2-minutecentrifugationThis technique is considered one of the fastestmost reliable and relatively easy ways to obtain plasmidDNAfrom cells Vigorous mixing during lysis and neutralizationphases can cause fragmentation of genomicDNA resulting incontamination with plasmid supernatant The purified DNAis suitable for less sensitive applications For more sensitiveapplications a purifying step is needed usually with spincolumns

26 Purification of Poly(A)+ RNA by Oligo(dT)-CelluloseChromatography Most of eukaryotic mRNA molecules pos-sess a polyadenylated (polyA) tail of about 250 nucleotidesat 31015840 end This provides foundation for a simple and easyway of RNA extraction through chromatographic techniquesThe basic mechanism of this method is that poly(A) RNAhybridizes with an oligo(dT)-cellulose matrix under high-salt conditions Eukaryotic mRNAs have a diverse range interms of size (from 05 kb to over 20 kb) and abundance(from fewer than 15 copies to over 20000 copies per cell)[37] Polyadenylated RNA with minimum 20 residues hasthe ability to attach to the oligo(dT)-cellulose matrix whichusually consists of 10ndash20 nucleotides [38] After washing outall the nonpolyadenylated RNAs a low-salt buffer is used todisrupt the oligo(dT)-poly(A) bond resulting in the elutionof poly(A) RNAs [39] The selection for poly(A) RNA canbe made in column or batch chromatography [1] being fastand yielding good RNA recovery Its drawback resides inthe fact that the method selects only mRNAs and naturallyexcludes important biological information present in otherRNAs such as miRNAs rRNAs and tRNAs

Table 2 summarizes the main advantages and disadvan-tages of the chemically driven methods discussed here

3 Solid-Phase Nucleic Acid Extraction

Solid-phase extraction (SPE) is one of the most efficient NAEtechniques available in the market [1 5] It is based on liquidand stationary phases which selectively separate the targetanalyte from the solution based on specific hydrophobic

polar andor ionic properties of both solute and sorbentThe chemistry between sorbent and analyte of interest is thebasis of this technique while ldquoweakrdquo chemical interactionssuch as van der Waals forces (nonpolar interactions) dipole-dipole interactions (polar interactions) and hydrogen bond-ing determine the retention mechanism in SPE

SPE methods can be divided into normalregular SPEreverse SPE and ion exchange SPE Every sorbent used inSPE has unique characteristics which give rise to a solutionfor a specific problem involved in extraction methods Agood example is acetonitrile which decreases the polar-ity of the solution and decreases the interaction of DNAmolecules with the stationary phase Normally reverse SPEuses polarmoderately mobile phase nonpolar stationaryphase and semi- or nonpolar analytes while normal SPEconsists of semi- to nonpolar mobile phase polar stationaryphase and polar analytes On the other hand ion exchangeSPE is based on electrostatic interaction of both sorbent andthe analyte of interest [40]

Solid-phase microextraction (SPME) is a relatively newdevelopment in solid-phase extraction technique introducedin 1990s by [41] being useful for various analytes includingliquid gaseous and solid matrices [42] Two importantsteps are involved in SPME (i) partitioning of analytes onfiber-coated extraction phase and (ii) handing over extractto separating instrument like gas chromatography wheredesorption takes place SPME is a rapid and easy to usetechnique and have good detection limit (parts per trillion)for specific compounds [43] Drawbacks of SPME includedifficulty in analyzing high molecular weight compoundssample carryover and the eventual shortage of commerciallyavailable stationary phases

31 Silica Matrices In 1979 it was found that silicates havehigh binding affinity for DNA under alkaline conditionsand increased salt concentration [44] Silica matrices haverevolutionized NAE procedures for both commercial as wellas research purposes Efficient and selective binding of NAto silica matrices is the hallmark of this fast and robustNA purification procedure [45] Silica matrices consist ofsilica material in the form of either gel or glass particle


(ie glass microfibers) [46] The mechanism involved inthis technique is the affinity between negatively charged NAand positively charged silica material resulting in selectivebinding of nucleic acids to the silica matrices while the restof the cell components and other chemicals are washed outSilica surface is covered by positive ions which enhancesthe binding of negatively charged DNA As a final step NAcan be eluted from silica matrix by any hyposmotic solutionsuch as nuclease-free water or buffers such as alkaline Tris-EDTA For RNA extraction chaotropic agents have a secondand very important task in denaturing RNases [47] Manymodifications have been made to the original proceduresuch as introduction of hydrated silicamatrix andmicrochip-based silica SPE [48] In this technique it is also noteworthythe role played by sodium ions in attracting the negativelycharged oxygen present in nucleic acidrsquos phosphate group andhelping NA become insoluble because of the phenomenonknown as ldquosalting outrdquo in the presence of high-salt conditionsand acidic pH [4]This technique provides high-purity DNAis easy to perform and also is able to reproduce quantitativelyas well as qualitatively Downside of this technique is beingunable to recover small fragments DNA efficiently as smallfragments binds tightly with the silica matrix [49]

32 Glass Particles Glass particles whether in powder aschromatography stationary phase or in microbeads formhave also been used for extraction of nucleic acids Chao-tropic salts are used to release the NA and allow bindingto common silicate glass flint glass and borosilicate glass(arranged as glass fiber filters) The basic principle of bindingof NA to glass particles is based on the adsorption affinityof the components present in the mixture (DNA proteinsetc) to the stationary phase of chromatography column(glass particles) Polar compounds such as DNA have ahigh tendency of attachment to polar stationary phase underspecific ionic strength [46]

33 Diatomaceous Earth Diatomaceous earth (DE) alterna-tively known as kieselguhr or diatomite is mainly composedof silica 80 to 90 (sometimes up to 95) alumina 2 to4 and hematite 05 to 2 [50] First described by Boomet al [51] this procedure is mainly based on the bindingof NA to a solid phase (such as DE) in the presence ofchaotropic agents followed by elution in water or low-saltbuffer NA binds to the silica present in DE following thesame principles of binding to silica matrices This procedurehas the advantage of reduced pipetting error shorter protocoltime and less number of steps for sample preparation beingused for plasmid as well as for single or double-strandednucleic acids [52] However this technique is not routinelyused because of comparably high cost

34 Magnetic Beads-Based Nucleic Acid Purification Mag-netic beads technology is one of the emerging strategies forextracting RNA and genomic plasmid and mitochondrialDNA The technique involves the separation of nucleic acidsfrom complex mixtures via complementary hybridization[53] In recent years functionalized magnetic particle orbeads have been coupled to suitable buffers systems for a

rapid and efficient extraction procedure [54] The lack ofcentrifugation steps that can produce shear forces and causebreaking of nucleic acids is thought to better maintain intactlonger fragments from genomic DNA Usually it is enoughto apply a magnet to the side of a vessel or tube containingthe sample mixed with the functionalized magnetic beadsand exclusively aggregate the target particles near the vesselwall The positive aspect of this technique is avoiding cen-trifugation steps as well as providing an alternative way forautomation of extraction procedures from a large numberof samples The extraction technique can be used in batchprocesses with a multitude of samples (blood tissues andothers) and is relatively easy to execute being one of thebest choices for automation high-throughput applicationsand high sample processivity [55 56] This method is alsosuitable for using in low technological environments becauseit is virtually equipment-free

35 Anion Exchange Material Just like silica matrices anionexchange resins are alsowidely used inDNAandRNAextrac-tion [57] Unlike silicate negative charge anion exchangeresin makes use of the positively charged diethylaminoethylcellulose (DEAE) to attract the negatively charged phosphateof nucleic acid So pH and salt concentration are theimportant aspects determining the binding or elution of NAto the anion exchange resin [58] Anion exchange has theadvantage of extracting very pure DNA as compared to silicaand the ability to reuse the resin upon renaturation Howeverthis method used high-salt concentration in the elution stepthus requiring desalting for downstream applications

36 Cellulose Matrix Absorbent cellulose-based paper is aninteresting matrix for nucleic acids purification and storageCellulose is a polymer of glucose and therefore highlyhydroxylated producing a polar attraction strong enough tobind nuclei acids under specific chemical conditions Thecommercially available FTA paper (Whatman Inc USA)or fast technology for analysis is an interesting example ofhow to purify and store DNA using cellulose FTA cards aresimply a thick layer of paper embedded with a proprietarymix of buffers detergents and chelating agents such as theubiquitous Tris pH 8 SDS and EDTA Once cells are spottedonto the paper the detergent lyses the membranes andEDTA chelates metal ions that are cofactors to nucleases alsopreventing the growth of contaminating organisms [59 60]Hence when dried nucleic acids are relatively well protectedfrom the environment especially due to the unavailability ofwater molecules In fact DNA extraction from dried bloodspotting has been successfully used for PCR-mediated humandiagnostics for more than 20 years [61ndash63]

Although FTA cards have many advantages regardingthe easiness of use and storage processing them to extractgood yields of nucleic acids might be more complicated thanexpected especially in diluted samples [64]

Table 3 summarizes the main advantages and disad-vantages of most commonly used solid-phase extractionmethods Table 4 gives examples of commercially availablekits using the methods described herein as well as givingtypical yields for NA extraction


Table 3 Summary of the advantages and disadvantages of solid-phase extraction methods

Material Molecule of affinity Advantage Disadvantage Reference

(1) Silica matrices DNA RNA High-purity DNA easy toperform and reproducible

Unable to recover smallDNA fragments one-timeuse

[52]

(2) Glass particles DNA protein Simple sensitive andreproducible

High cost requirement ofequipment [1 52]

(3) Diatomaceous earth DNA RNAReduced pipetting errorshorter protocol (less timeand steps)

High cost [53]

(4) Magnetic beads DNA RNANo centrifugation bestchoice for automationvirtually equipment-free

Interference in PCRamplification [56]

(5) Anion exchange material DNA RNA Reusable resins Presence of high-saltconcentrations [58]

(6) Cellulose matrix DNA RNA Easy to use and storage Extraction protocols beingcomplex and prone to error [60]

Table 4 Examples of commercially available kits applying each extraction method and typical yields for distinct samples

Method Commercial availability Sample origin Typical yield ReferenceGSCN-phenol-chloroformextraction

TRIzol reagent (egInvitrogen) Mammalian cells (106 cells) Epithelial cells 8ndash15 120583g

fibroblasts 5ndash7 120583g [65]

Alkaline extraction Plasmid Maxi Kit (egQiagen) Cultured bacteria (25 L) Up to 500 120583g of plasmid

DNA [66ndash68]

CTABNucleoSpin 8 Plant andNucleoSpin 96 Plant II (egClontech)

Plant material 20ndash100mgplant tissue (wet weight) 1ndash30 120583g [69 70]

Silica matrices QIAamp DNAmini kit (egQiagen)

Blood (200 120583l) buffy coat(200120583l) or 106 cells

4ndash12120583g DNA (blood)25ndash50 120583g DNA (buffy coat)15ndash20 120583g DNA (cells)

[71 72]

Magnetic bead Agencourt DNAdvance Kit(eg Beckman Coulter)

Mammalian tissues (25mgof sample) 18ndash35 120583g DNA [73]

Anion exchange materialPureLinkHiPure PlasmidDNA Purification Kits (egInvitrogen)

Culture bacteria midipreps(15ndash25mL) or maxiprep(up to 200mL)

350 120583g for midipreps850 120583g for maxipreps [74]

Diatomaceous EarthQuantum Prep PlasmidPurification Kits (egBio-Rad)

Culture bacteria (1-2mLliquid culture) Up to 40 120583g [75]

Cellulose matrix FTA cards (eg Whatman) 8 times 2mm punches 1ndash5 120583g (plant)1ndash3 120583g (dried blood spots) [61ndash63 76 77]

4 Devices Used in Extraction Methods

41 Spin Columns The binding element in spin-column sys-tems is usually composed of glass particles or powder silicamatrices diatomaceous earth and ion exchange carriersNucleic acid binding is thus optimized with specific buffersolutions and extremely precise pH and salt concentrations[4] Column-based NAE is one of the best techniques amongthe options available playing a vital role in ion exchangemethods as it provides a robust stationary phase for a rapidand reliable buffer exchange and thus NAE This method isfast and reproducible and its main drawback is the need fora small centrifuge as equipment requirement

42 Beads or Magnetic Beads Magnetic particle or beads arethe first option to eliminate centrifuge-dependent steps inthe extraction process Magnetic beads make use of differentligands such as antibodies antigens oligonucleotides oraptamers which bind specifically to its target in sampleThe first magnetic particle used for extraction consisted ofan iron-oxide core covered by functional carboxylic groupwhich then binds DNA or RNA [81] Since then manymodifications have been made using different surface func-tional group such as sulphate amino and hydroxyl groups[82] Besides these functional groups preactivated magneticbeads with different functional groups are available such astosyl or epoxy groups [78 83] Magnetic beads activated


Table 5 Summary of available devices used in nucleic acid extraction protocols


(1) Spin columns DNA RNA Fast reproducible

Aerosolscross-contaminationinfrastructure andequipment required

[49]

(2) Beads or magnetic beads DNA RNASimple to use highautomation potentialequipment-free process

Labor intensive [78]

(3) Automation (liquid handlingrobots) DNA RNA

Precise manipulation ofsample and reagentsreducing losses and cost

High cost [79]

(4) Microfluidics andldquolab-on-a-chiprdquo cartridges DNA RNA Sensitive and specific Incompatibility of common

NAE chemicals [80]

with protein A protein G or streptavidin are commerciallyavailable Magnetic bead separation presents many benefitsover centrifuge-dependent extraction process by allowing anequipment-free process Equipment-free separation of NA isalso possible with nonmagnetic beads where the beads aretrapped inside plastic bubble pipets [84]

43 Automation (Liquid Handling Robots) The increase ingrowth of diagnostic tests and patient numbers highlightsthe need for automation in life sciences [85] To fulfill thisdemand various automated devices have been developed andintroduced in the market The most successful examples arethe automated liquid handling robots which are routinelyused in many life science and clinical analysis laboratoriesfor dispensing precise amount of sample reagents or otherliquids to designated containers Because of this technologyit is now possible to handle many samples simultaneouslywith precision and rapidity In addition barcode readersare an integral part of such equipment allowing for easytraceability of samples and results Fully automated NAEprotocols have been developed for such equipment usingeither solid-phase ormagnetic beadsmethods [79] Howeverhigh sample processivity is a positive aspect of automationwhile maintaining the sensitivity can be compromised aslow-copy NA targets might be lost [86] Small versions ofthese robots are available and could be useful in laboratorysettings with minimal infrastructure Liquid handling robotscertainly have a niche in life sciences and clinical laboratoriesbut not as POC devices

44 Microfluidics and ldquoLab-on-a-Chiprdquo Cartridges Nucleicacid-based detection (NAT) is preferable compared toimmunoassay-based detection because of sensitivity andspecificity but NAT-based diagnosis requires complex infras-tructure sophisticated machinery and trained personnel Toovercome this hurdle microfluidic chips have been designedand produced carrying on inner chambers all necessaryreagents for molecular based tests as a part of POC-Dxstrategy Usually microfluidic chips (or ldquolab-on-a-chiprdquo car-tridges) rely on solid phase extraction for NA isolation in theform of either membranes or beads [44 87]

The union of automation with the need for miniaturiza-tion in POC devices led to the development of cartridgesthat perform one or several biological reactions in a closedcontainer These reactions comprise most of the currentmolecular biology methods such as NAE amplificationand identification as well as serological signatures analysesOne of the greatest examples of a microfluidic cartridgealthough not POC is the milestone related to diagnosis ofMycobacterium tuberculosis (Mtb) achieved by the platformGeneXpert MTBRIF [80] Specific cellsspores are selectedthrough filtration followed by a lysing step (sonification)Microtubes pumps and rotary drives transfer liquids intothe specific cartridge chambers where washing purificationand concentration of nucleic acids take place The next stepis the movement of the extracted NA to a reaction chamberwhere real-time PCRhappens [88] A recent systematicmeta-analysis study reviewed hundreds of papers concluded thatGeneXpert was the most cost-effective strategy for POC-DxofMtb although its performance was evaluated solely in clin-ics and primary care centers [89] However it is undisputedthat GeneXpert is a breakthrough in NA testing

The FilmArray 20 system (BioFire Diagnostics LLCSalt Lake City USA) is a multiplexed PCR system thatincorporates specimen processing NA amplification anddetection in a specialized pouch Specific pouches are usedto amplify different targets present in the sample usingNested PCR followed by real-time PCR with chemistry-based detection The software then automatically generatesidentification reports using DNA melting analysis based onspecific control reactions or a melting curve database ofknown sequences

Table 5 presents a summary of the devices available mostcommonly used in NAE protocols

5 Limitations for Implementation ofExtraction Protocols in Portable Devices

A major obstruction for the development of a complete andeasy-to-use solution for POC-Dx is the integration of samplepreparation protocols into the portable devices Removinginterferents and extracting the target molecules are no trivial


Table 6 Chemical compatibility of various chemicals used in nucleic acid extraction procedures and plastic polymers commonly usedin microfabrication (PDMS polydimethylsiloxane PMMA polymethylmethacrylate PS polystyrene PC polycarbonates) Informationwas gathered from multiple sources such as the following vendorrsquos websites httpwwwpermselectcom httpwwwlabicomcomczhttpswwwcoleparmercoukChemical-Resistance

ChemicalsPlastic polymer PDMS PMMA PS PCEthanol Good Good Not compatible ExcellentPhenol 10 Not compatible Not compatible Not compatible Not compatibleChloroform Not compatible Not compatible Not compatible Not compatibleDetergents Excellent mdash mdash mdashUrea Good Excellent Excellent ExcellentGuanidinium thiocyanate mdash Good mdash ExcellentMethyl alcohol Excellent Good Good FairAlcohols isopropyl Excellent Excellent Excellent FairAlcohols ethyl Good Good Good ExcellentAlcohols amyl Not compatible Excellent Good GoodHCl 37 Not compatible mdash mdash mdashHCl 20 Good Good Good Good

task especially due to the vast differences among samplematrices as well as characteristics of the target analytesWhile NAE protocols are well established in the laboratoryand many advances have been made since the inceptionof microfluidic Dx devices commercial availability of thesedevices is still rare [90] Excellent reviews are availablediscussing the technical difficulties as well as the obstacles forimplementation and acceptance of new tests based on newtechnologies [90ndash94]

Several organic chemicals routinely used in molecularbiology can react with the plasticmaterials commonly used inPOC cartridgesdevices whichmakes difficult for some poly-mers to sustain their initial mechanical and physicochemicalproperties One of properties paramount to the performancecharacteristics of the plastic materials is chemical inertnessthat is the material to which the active substance of inter-est will be in contact with will not interact and generateundesirable products generally classified as extractable orleachable [95] Toxicological or functional studies oftenreplace extraction and interaction studies which would benecessary to determine the levels of extractable or leach-able products under a given environmental condition Suchreplacement is acceptable although not ideal because thebiological assessment performed for toxicological studiesshould include basic extractioninteraction evaluations [95]Studies of structural properties of glassy polymers suchas the commonly used thermoplastics polycarbonate (PC)and polymethylmethacrylate (PMMA) correlate the polymersolubility when exposed to several solvents to the extentof stress cracking [96] An advantage of PMMA is its highoptical transparency into the ultraviolet range while PCoffers a compatibility with a wider range of solvents and ahigher glass transition temperature well suited to applicationssuch as polymerase chain reaction for NA amplification[97] However neither of these is good enough to be usedwith the chemicals routinely used for NAE For examplePMMA cannot be cleaned by strong solvents such as acetone

or methanol because these chemicals would significantlydamage its surface and decrease transparency [97] (Table 6)

Some chemicals have the potential to affect polymerrsquoscolor surface appearance flexibility and mass generatingextractableleachable products that must be evaluated Thesechanges can happen due to several physicochemical reac-tions such as (i) chemical interaction with polymer chainwhich can disturb their structure and result in depolymeriza-tion (ii) physical interaction that is adsorption of chemicalsinto the plastics which results in swelling and softening or(iii) stress-associated cracking may happen due to the stress-cracking agents such as plasticizers or adhesives used duringthe manufacturing of polymer parts or even detergents oroils used during themolecular biology processes [98] Table 6lists the effect of the chemicals most commonly used in NAEon the plastics most commonly used for microfabricationof microdevices Alterations induced by any chemical asminor as it seems need to be thoroughly evaluated Inextreme cases chemicals must be substituted such as thatof ethanolisopropanol Ethanolisopropanol storage in car-tridges is also problematic because of its volatility flamma-bility and potential to leak Such chemical properties makealcohols a substance highly regulated by the International AirTransport Association (IATA) Therefore substitutes such asdiethylene glycol monoethyl ether (DGME) and diethyleneglycol monoethyl ether acetate (DGMEA) [99] must betested and validated

Finally yet importantly there is concern about the volumeof sample needed to obtain a meaningful results [100]Because the volume of buffers and therefore of harshchemicals used for cell lysis is directly proportional to thevolume of the sample POC-Dx tests aremost useful in illnesswhere the pathogen is present in higher counts such as virusand most bacterial infections Parasitic infections howeverpresent a challenge to POC-Dx because parasite loads can getvery close to the limits of detection of the techniques used[101] thus greatly affecting the availability of target NA in the


sampleThe volume of the reagents is also important to assureproper mixing of solutions without the common laboratoryinstruments because small volumes are easier to homogenize[102]

6 Challenges for Implementation in POCDiagnostic Tests

Lessons learned from previous attempts in developing diag-nostic tests have taught us that availability of the best possiblePOC-Dx test is not enough Its implementation is also veryimportant and often underestimated since only few diseaseshave a validated POC-Dx such as HIV or malaria [103 104]Implementation should be considered during the develop-ment phase of the POC-Dx so that end-users are identifiedtheir level of experience is assessed and the developing test isused at the right lab tier [92]

The major features of POC tests are described by theWHO acronym ASSURED affordable sensitive specificuser-friendly equipment-free and deliverable to end-users[103] The main idea is to provide low cost and timelyeffective healthcare to the patient and quick decision mak-ing for healthcare providers One platform which seemsto have the potential to meet the ASSURED criteria ismicrofluidic paper-based analytical devices (120583PADs) whichprovide a robust affordable equipment-free and multiplexfacility [105ndash107] Paper-based devices are abundant eitherdirectly operating or directing the biochemical serologicalor nucleic acid reactions [106 107] Because they are easilymanipulated to attach recognition molecules (antibodiesenzymes proteins nucleic acids etc) 120583PADs devices havebeen very successful in several areas of biological researchsuch as biochemical analysis of blood or urine detection ofpathogenrsquos nucleic acids detection of drugs or environmentalcontamination120583PADs can also be designed for direct sensingthe target molecule by using nanotechnologies such asmicroelectromechanical systems field effector transistorsor nanocantilevers However since describing each of theavailable 120583PADs is beyond the scope of our review the readeris directed to other available texts on the subject [106ndash109]Unlike protein or metabolite-based POC tests one of themajor challenges for nucleic acid-based POC tests is the needto consolidate three distinct protocol procedures into a singledevice (1) nucleic acid extraction (2) amplification and (3)detection Development of a nucleic-based testing device thatis specific sensitive portable and relatively easy to operatehas presented several challenges that have been elegantlyreviewed elsewhere [90] Below we discuss the challengesstrictly related to NAE for POC-Dx

Development of an ideal NAE method for POC isimpaired by many factors and researchers are still in questfor a suitable solution At present solid-phase extraction[110] and magnetic beads [54] are the primary choices forNAE in POC-Dx devices However neither method is yetgood enough for widespread implementation in POC-Dxmethods Solid-phase extraction depends on centrifugationwhile magnetic beads require an external magnet source formixing In this aspect magnetic beads are favored because

implementation of magnetic stirring in POC-Dx devices issomewhat easier than implementation of separation throughstationary membranes Although both rely on the use ofchaotropic reagents for lysing cells and releasing the NAfrom the scaffold and structural nucleic proteins washingsteps are more efficient in beads-based methods The mainchallenges in implementingmolecular biology-based systemsin resource-constrained areas are the high cost of instrumentand reagents as well as lack of reliable infrastructure and con-tinuous maintenance support and temperature maintenancedevices [88] Proper disposal of biological waste generatedby medical tests is also a concern not to mention thatsome waste is chemical and requires special treatment beforedisposal (eg guanidine thiocyanate) [92]

7 Conclusion

After almost 150 years after the first successful isolation ofDNA by Friedrich Miescher nucleic acids are now centralto obtaining biological information in areas as distinct asspecimensrsquo identification for conservational purposes to therealms of personalized medicine and pharmacogenomicsProtocols and devices used for NAE have evolved fromthiocyanate-phenol-chloroform manual techniques to user-friendly column-technology and automated platforms butno general gold-standard method has yet been establishedThis review analyzed the working principle of each availablemethod as well as their advantages and disadvantages Thetake-home message is that each application has specificcharacteristics which should then guide each researcher tothe most suitable method

Although molecular biology techniques are sensitive andaccurate methods they require a rather well establishedlaboratory setting and expensive instruments as well asskilled personnel to run the tests and analyze the resultswhich are not always available In the last years lab-on-chip technology has brought the promise of taking themanagement of biological information where it is neededsuch as low-resource settings a doctorrsquos clinic or a hospitalpatient bedside However although progress has been madeseveral obstacles still hamper the use of NAE protocols inPOC-Dx tests as it can be seen by the lownumber of productsusing lab-on-chip technology Overcoming the challengesand limitations of NAE protocols will greatly increase the useof molecular biology techniques and thus increase the overallquality of life of the general population by providing access tobetter diagnostic tests

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

The authors are grateful to Dr Fabrıcio K Marchini and DrAdriana C S Umaki for critical reading of the manuscript


References

[1] S C Tan and B C Yiap ldquoDNA RNA and protein extractionthe past and the presentrdquo Journal of Biomedicine and Biotechnol-ogy vol 2009 Article ID 574398 10 pages 2009

[2] K Doyle The Source of Discovery Protocols and ApplicationsGuide PROMEGA Madison Madison Wis USA 1996

[3] AM Lesk ldquoWhy doesDNA contain thymine andRNAuracilrdquoJournal of Theoretical Biology vol 22 no 3 pp 537ndash540 1969

[4] D Chacon-cortes L Griffiths and D Chacon-Cortes ldquoMeth-ods for extracting genomic DNA from whole blood sam-ples current perspectivesrdquo Journal of Biorepository Science forApplied Medicine vol 2 pp 1ndash9 2014

[5] C W Price D C Leslie and J P Landers ldquoNucleic acidextraction techniques and application to the microchiprdquo Lab ona ChipmdashMiniaturisation for Chemistry and Biology vol 9 no17 pp 2484ndash2494 2009

[6] S Goldberg ldquoMechanicalphysical methods of cell distributionand tissue homogenizationrdquoMethods in Molecular Biology vol1 no 424 pp 3ndash22 2008

[7] S Mitra ldquoSample preparation techniques in analytical chem-istryrdquo in Chemical Analysis - A Series of Monographs onAnalytical Chemistry and Its Applications J D WinefordnerEd vol 162 JohnWiley amp Sons Inc Hoboken NJ USA 2003

[8] D Burden ldquoGuide to the Disruption of Biological SamplesrdquoRandom Prim vol 25 no 12 p 25 2012

[9] KayaG C Dale IMaudlin andKMorgan ldquoA novel procedurefor total nucleic acid extractionfrom small numbers of eimeriaspecies oocystsrdquoThe Turkish Society for Parasitology vol 3 no31 pp 180ndash183 2007

[10] S Peternel and R Komel ldquoIsolation of biologically activenanomaterial (inclusion bodies) from bacterial cellsrdquoMicrobialCell Factories vol 9 article 66 2010

[11] E Rodrıguez-Carmona O Cano-Garrido J Seras-FranzosoA Villaverde and E Garcıa-Fruitos ldquoIsolation of cell-freebacterial inclusion bodiesrdquo Microbial Cell Factories vol 9article 71 2010

[12] P Prabakaran and A D Ravindran ldquoA comparative studyon effective cell disruption methods for lipid extraction frommicroalgaerdquo Letters in Applied Microbiology vol 53 no 2 pp150ndash154 2011

[13] O Salazar and J A Asenjo ldquoEnzymatic lysis of microbial cellsrdquoBiotechnology Letters vol 29 no 7 pp 985ndash994 2007

[14] S Flahaut J Frere P Boutibonnes andY Auffray ldquoComparisonof the bile salts and sodium dodecyl sulfate stress responses inEnterococcus faecalisrdquo Applied and Environmental Microbiol-ogy vol 62 no 7 pp 2416ndash2420 1996

[15] R G Harrison P Todd S R Rudge andD P Petrides Biosepa-rations Science and Engineering Oxford University press 2003

[16] D Liu X-A Zeng D-W Sun and Z Han ldquoDisruption andprotein release by ultrasonication of yeast cellsrdquo Innovative FoodScience and Emerging Technologies vol 18 pp 132ndash137 2013

[17] M Klimek-OchabM Brzezinska-Rodak E Zymanczyk-DudaB Lejczak and P Kafarski ldquoComparative study of fungalcell disruption-scope and limitations of the methodsrdquo FoliaMicrobiologica vol 56 no 5 pp 469ndash475 2011

[18] J M Greenly and J W Tester ldquoUltrasonic cavitation fordisruption of microalgaerdquo Bioresource Technology vol 184 pp276ndash279 2015

[19] F Bunge M Pietzsch R Muller and C Syldatk ldquoMechanicaldisruption of Arthrobacter sp DSM 3747 in stirred ball mills

for the release of hydantoin-cleaving enzymesrdquo Chemical Engi-neering Science vol 47 no 1 pp 225ndash232 1992

[20] TA BrownGene cloningampDNAAnalysis An Introduction Sixthedition Blackwell Publishing 6th edition 2010

[21] L J Cseke P B Kaufman A Kirakosyan and M V WestfallldquoMolecular and Cellular Methods in Biology and Medicinerdquo2011

[22] S M Carr and O M Griffith ldquoRapid isolation of animalmitochondrial DNA in a small fixed-angle rotor at ultrahighspeedrdquo Biochemical Genetics vol 25 no 5-6 pp 385ndash390 1987

[23] K Nath J W Sarosy J Hahn and C J Di Como ldquoEffectsof ethidium bromide and SYBR() Green I on different poly-merase chain reaction systemsrdquo Journal of Biochemical andBiophysical Methods vol 42 no 1-2 pp 15ndash29 2000

[24] T Ohta S-I Tokishita and H Yamagata ldquoEthidium bromideand SYBR Green I enhance the genotoxicity of UV-irradiationand chemical mutagens in E colirdquoMutation ResearchmdashGeneticToxicology and Environmental Mutagenesis vol 492 no 1-2 pp91ndash97 2001

[25] P Chomczynski and N Sacchi ldquoSingle-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextractionrdquo Analytical Biochemistry vol 162 no 1 pp 156ndash1591987

[26] A Ullrich J Shine J Chirgwin et al ldquoRat insulin genesconstruction of plasmids containing the coding sequencesrdquoScience vol 196 no 4296 pp 1313ndash1319 1977

[27] J M Chirgwin A E Przybyla R J MacDonald and W JRutter ldquoIsolation of biologically active ribonucleic acid fromsources enriched in ribonucleaserdquo Biochemistry vol 18 no 24pp 5294ndash5299 1979

[28] P Chomczynski andN Sacchi ldquoThe single-stepmethod of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextraction twenty-something years onrdquoNature Protocols vol 1no 2 pp 581ndash585 2006

[29] L Meng and L Feldman ldquoA rapid TRIzol-based two-stepmethod for DNA-free RNA extraction from Arabidopsissiliques and dry seedsrdquo Biotechnology Journal vol 5 no 2 pp183ndash186 2010

[30] M JMurnane and SW Tam ldquoIsolation and characterization ofRNA from snap-frozen tissues and cultured cellsrdquo The Journalof Nutritional Biochemistry vol 3 no 5 pp 251ndash260 1992

[31] C F TerryNHarris andHC Parkes ldquoDetection of geneticallymodified crops and their derivatives critical steps in samplepreparation and extractionrdquo Journal of AOAC International vol85 no 3 pp 768ndash774 2002

[32] T Demeke and G R Jenkins ldquoInfluence of DNA extractionmethods PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traitsrdquoAnalytical andBioanalytical Chemistry vol 396 no 6 pp 1977ndash1990 2010

[33] P S Walsh D A Metzger and R Higuchi ldquoChelex 100 as amedium for simple extraction of DNA for PCR-based typingfrom forensic materialrdquo BioTechniques vol 10 no 4 pp 506ndash513 1991

[34] K Phillips N McCallum and L Welch ldquoA comparison ofmethods for forensic DNA extraction Chelex-100 and theQIAGEN DNA Investigator Kit (manual and automated)rdquoForensic Science International Genetics vol 6 no 2 pp 282ndash285 2012

[35] C Schrader A Schielke L Ellerbroek and R Johne ldquoPCRinhibitorsmdashoccurrence properties and removalrdquo Journal ofApplied Microbiology vol 113 no 5 pp 1014ndash1026 2012


[36] H C Bimboim and J Doly ldquoA rapid alkaline extraction pro-cedure for screening recombinant plasmid DNArdquoNucleic AcidsResearch vol 7 no 6 pp 1513ndash1523 1979

[37] S Bryant and D L Manning ldquoIsolation of mRNA by affinitychromatographyrdquo The Nucleic Acid Protocols Handbook pp 9ndash11 2000

[38] J M Walker Nucleic Acids vol 2 Humana Press New JerseyNJ USA 1984

[39] J Sambrook and W Russel Molecular Cloning 3-Volume SetA Laboratory Manual vol 3 Cold Spring Harboc LaboratoryPress 2000

[40] M Biziuk ldquoSolid Phase Extraction TechniquemdashTrendsrdquo PolishJournal of Environmental Studies vol 15 no 5 pp 677ndash6902006

[41] C LArthur and J Pawliszyn ldquoSolid phasemicroextractionwiththermal desorption using fused silica optical fibersrdquo AnalyticalChemistry vol 62 no 19 pp 2145ndash2148 1990

[42] S Merkle K Kleeberg and J Fritsche ldquoRecent developmentsand applications of solid phasemicroextraction (SPME) in foodand environmental analysismdasha reviewrdquoChromatography vol 2no 3 pp 293ndash381 2015

[43] A T Nielsen and S Jonsson ldquoTrace determination of volatilesulfur compounds by solid-phase microextraction and GC-MSrdquo Analyst vol 127 no 8 pp 1045ndash1049 2002

[44] B Vogelstein and D Gillespie ldquoPreparative and analyticalpurification of DNA from agaroserdquo Proceedings of the NationalAcademy of Sciences vol 76 no 2 pp 615ndash619 1979

[45] K-H Esser W H Marx and T Lisowsky ldquomaxXbond Firstregeneration system for DNA binding silica matricesrdquo NatureMethods vol 3 no 1 pp 2005-2006 2006

[46] V V Padhye C York and B Adam ldquoNucleic acid purificationon silica gel and glass mixturerdquo in United States patent US5658548 Promega Corporation 1997

[47] A I Mutin and J B Christopher ldquoRNA isolation from yeastusing silica matricesrdquo Journal of Biomolecular Techniques vol16 no 4 pp 316-317 2005

[48] L A Christel K Petersen W McMillan and M A NorthrupldquoRapid automated nuleic acid probe assays using siliconmicrostructures for nucleic acid concentrationrdquo Journal ofBiomechanical Engineering vol 121 no 1 pp 22ndash27 1999

[49] M R Green and J SambrookMolecular Cloning vol 1 2012[50] L E Antonides ldquoDiatomiterdquo pp 1ndash6 1997[51] R Boom C J Sol M M Salimans C L Jansen P M

Wertheim-van Dillen and J van der Noordaa ldquoRapid andsimple method for purification of nucleic acidsrdquo Journal ofClinical Microbiology vol 28 no 3 pp 495ndash503 1990

[52] K Koo P M Foegeding and H E Swaisgood ldquoIsolation ofRNA and DNA fragments using diatomaceous earthrdquo Biotech-nology Techniques vol 12 no 7 pp 549ndash552 1998

[53] M J Archer B Lin Z Wang and D A Stenger ldquoMagneticbead-based solid phase for selective extraction of genomicDNArdquo Analytical Biochemistry vol 355 no 2 pp 285ndash2972006

[54] S M Azimi G Nixon J Ahern andW Balachandran ldquoAmag-netic bead-based DNA extraction and purification microfluidicdevicerdquoMicrofluidics andNanofluidics vol 11 no 2 pp 157ndash1652011

[55] S Berensmeier ldquoMagnetic particles for the separation andpurification of nucleic acidsrdquoAppliedMicrobiology and Biotech-nology vol 73 no 3 pp 495ndash504 2006

[56] M Franzreb M Siemann-Herzberg T J Hobley and O R TThomas ldquoProtein purification using magnetic adsorbent parti-clesrdquo Applied Microbiology and Biotechnology vol 70 no 5 pp505ndash516 2006

[57] D B Seligson and E J Shrawder United States Patent [19]1Patent Number 5387720 4935342 1995

[58] Qiagen ldquoGenomic DNA Handbookrdquo 2001[59] L M Smith and L A Burgoyne ldquoCollecting archiving and

processing DNA from wildlife samples using FTA databasingpaperrdquo BMC Ecology vol 4 article 4 2004

[60] L A Burgoyne ldquoSolid medium and method for DNA storagerdquoTech Rep 5496562 1996

[61] S A Cassol N Lapointe T Salas et al ldquoDiagnosis of verticalHIV-1 transmission using the polymerase chain reaction anddried blood spot specimensrdquo Journal of Acquired ImmuneDeficiency Syndromes vol 5 no 2 pp 113ndash119 1992

[62] S A Cassol S Read B G Weniger et al ldquoDried blood spotscollected on filter paper an international resource for the diag-nosis and genetic characterization of human immunodeficiencyvirus type-1rdquo Memorias do Instituto Oswaldo Cruz vol 91 no3 pp 351ndash358 1996

[63] E-H Choi S K Lee C Ihm and Y-H Sohn ldquoRapid DNAextraction from dried blood spots on filter paper potentialapplications in biobankingrdquo Osong Public Health and ResearchPerspectives vol 5 no 6 pp 351ndash357 2014

[64] E Milne F M Van Bockxmeer L Robertson et al ldquoBuccalDNA collection comparison of buccal swabs with FTA cardsrdquoCancer Epidemiology Biomarkers and Prevention vol 15 no 4pp 816ndash819 2006

[65] A B Hummon S R Lim M J Difilippantonio and TRied ldquoIsolation and solubilization of proteins after TRIzolextraction of RNA and DNA from patient material followingprolonged storagerdquo BioTechniques vol 42 no 4 pp 467ndash4722007

[66] V S Somvanshi R E Sloup J M Crawford et al ldquoA singlepromoter inversion switches photorhabdus between pathogenicand mutualistic statesrdquo Science vol 336 no 6090 pp 88ndash932012

[67] S Klinge F Voigts-Hoffmann M Leibundgut S Arpagausand N Ban ldquoCrystal structure of the eukaryotic 60S ribosomalsubunit in complex with initiation factor 6rdquo Science vol 334no 6058 pp 941ndash948 2011

[68] A Antonic E S Sena J S Lees et al ldquoStem cell transplantationin traumatic spinal cord injury a systematic review and meta-analysis of animal studiesrdquo PLoS Biology vol 11 no 12 ArticleID e1001738 2013

[69] A M Niewiadomska and R J Gifford ldquoThe extraordinaryevolutionary history of the reticuloendotheliosis virusesrdquo PLoSBiology vol 11 no 8 Article ID e1001642 2013

[70] C Varenhorst S James D Erlinge et al ldquoGenetic variation ofCYP2C19 affects both pharmacokinetic and pharmacodynamicresponses to clopidogrel but not prasugrel in aspirin-treatedpatients with coronary artery diseaserdquo European Heart Journalvol 30 no 14 pp 1744ndash1752 2009

[71] S J Rulli Jr J Mirro S A Hill et al ldquoInteractions of murineAPOBEC3 and human APOBEC3G with murine leukemiavirusesrdquo Journal of Virology vol 82 no 13 pp 6566ndash6575 2008

[72] S Ogino T Kawasaki K Nosho et al ldquoLINE-1 hypomethy-lation is inversely associated with microsatellite instabilityand CpG island methylator phenotype in colorectal cancerrdquoInternational Journal of Cancer vol 122 no 12 pp 2767ndash27732008


[73] MG Fischer andC A Suttle ldquoA virophage at the origin of largeDNA transposonsrdquo Science vol 332 no 6026 pp 231ndash234 2011

[74] H Nie and C-H Wang ldquoFabrication and characterization ofPLGAHAp composite scaffolds for delivery of BMP-2 plasmidDNArdquo Journal of Controlled Release vol 120 no 1-2 pp 111ndash1212007

[75] N J Johnston J C De Azavedo J D Kellner and D ELow ldquoPrevalence and characterization of the mechanisms ofmacrolide lincosamide and streptogramin resistance in iso-lates of Streptococcus pneumoniaerdquo Antimicrobial Agents andChemotherapy vol 42 no 9 pp 2425-2426 1998

[76] C S Siegel F O Stevenson and E A Zimmer ldquoEvaluation andcomparison of FTA Card and CTAB DNA extraction methodsfor non-agricultural taxardquo Applications in Plant Sciences vol 5no 2 p 1600109 2017

[77] C G Molteni L Terranova A Zampiero C Galeone NPrincipi and S Esposito ldquoComparison of manual methods ofextracting genomic DNA from dried blood spots collected ondifferent cards Implications for clinical practicerdquo InternationalJournal of Immunopathology and Pharmacology vol 26 no 3pp 779ndash783 2013

[78] J G Bruno and J L Kiel ldquoUse of magnetic beads in selectionand detection of biotoxin aptamers by electrochemilumines-cence and enzymatic methodsrdquo BioTechniques vol 32 no 1 pp178ndash183 2002

[79] G Rule M Chapple and J Henion ldquoA 384-well solid-phaseextraction for LCMSMS determination of methotrexate andits 7-hydroxy metabolite in human urine and plasmardquo Analyti-cal Chemistry vol 73 no 3 pp 439ndash443 2001

[80] C C Boehme P Nabeta D Hillemann et al ldquoRapid moleculardetection of tuberculosis and rifampin resistancerdquo The NewEngland Journal ofMedicine vol 363 no 11 pp 1005ndash1015 2010

[81] T Hawkins ldquoDNA Purification and Isolation using magneticparticlesrdquo US5705628A 1998

[82] N M Adams H Bordelon K-K A Wang L E Albert DW Wright and F R Haselton ldquoComparison of three magneticbead surface functionalities for RNA extraction and detectionrdquoACS Applied Materials and Interfaces vol 7 no 11 pp 6062ndash6069 2015

[83] S Centi S Laschi andMMascini ldquoStrategies for electrochem-ical detection in immunochemistryrdquoBioanalysis vol 1 no 7 pp1271ndash1291 2009

[84] D R Pawlowski and R J Karalus ldquoElectricity-free sequentialnucleic acid and protein isolationrdquo Journal of Visualized Exper-iments no 63 Article ID e4202 2012

[85] R S Markin and S A Whalen ldquoLaboratory automationtrajectory technology and tacticsrdquo Clinical Chemistry vol 46no 5 pp 764ndash771 2000

[86] S A Thatcher ldquoDNARNA preparation for molecular detec-tionrdquo Clinical Chemistry vol 61 no 1 pp 89ndash99 2015

[87] M G Mauk C Liu M Sadik and H H Bau ldquoMicrofluidicdevices for nucleic acid (NA) isolation isothermal NA ampli-fication and real-time detectionrdquo Mobile Health TechnologiesMethods and Protocols pp 15ndash40 2015

[88] M A Dineva L Mahilum-Tapay and H Lee ldquoSample prepa-ration a challenge in the development of point-of-care nucleicacid-based assays for resource-limited settingsrdquo Analyst vol132 no 12 pp 1193ndash1199 2007

[89] F Drobniewski M Cooke J Jordan et al ldquoSystematic reviewmeta-analysis and economicmodelling of molecular diagnostictests for antibiotic resistance in tuberculosisrdquo HealtH Technol-ogy Assessment vol 19 no 34 pp 1ndash188 2015

[90] F Cui M Rhee A Singh and A Tripathi ldquoMicrofluidicsample preparation for medical diagnosticsrdquo Annual Review ofBiomedical Engineering vol 17 pp 267ndash286 2015

[91] D Huckle ldquoThe impact of new trends in POCTs for companiondiagnostics non-invasive testing and molecular diagnosticsrdquoExpert Review of Molecular Diagnostics vol 15 no 6 pp 815ndash827 2015

[92] S Wang M A Lifson F Inci L-G Liang Y-F Sheng and UDemirci ldquoAdvances in addressing technical challenges of point-of-care diagnostics in resource-limited settingsrdquo Expert Reviewof Molecular Diagnostics vol 16 no 4 pp 449ndash459 2016

[93] V Hardy M Thompson W Alto et al ldquoExploring the barriersand facilitators to use of point of care tests in family medicineclinics in the United Statesrdquo BMC Family Practice vol 17 no 1pp 1ndash8 2016

[94] V Busin B Wells M Kersaudy-Kerhoas W Shu and S T GBurgess ldquoOpportunities and challenges for the application ofmicrofluidic technologies in point-of-care veterinary diagnos-ticsrdquo Molecular and Cellular Probes vol 30 no 5 pp 331ndash3412016

[95] D Jenke ldquoEvaluation of the chemical compatibility of plasticcontact materials and pharmaceutical products safety con-siderations related to extractables and leachablesrdquo Journal ofPharmaceutical Sciences vol 96 no 10 pp 2566ndash2581 2007

[96] G A Bernier and R P Kambour ldquoThe role of organic agentsin the stress crazing and cracking of poly(26-dimethyl-14-phenylene oxide)rdquo Macromolecules vol 1 no 5 pp 393ndash4001968

[97] S Yang and D L DeVoe ldquoMicro fluidic device fabrication bythermoplastic hot-embossingrdquo Methods in Molecular Biologyvol 949 pp 115ndash123 2013

[98] L M Robeson ldquoEnvironmental stress cracking a reviewrdquoPolymer Engineering and Science vol 53 no 3 pp 453ndash4672013

[99] R Himmelreich and S Werner ldquoMethods for Isolation andpurifying nucleic acidsrdquo Article ID 0224419 US 20110224419A1 2011

[100] E J Fong C Huang J Hamilton et al ldquoAmicrofluidic platformfor precision small-volume sample processing and its use tosize separate biological particles with an acoustic microdevicerdquoJournal of Visualized Experiments vol 2015 no 105 Article IDe5305 2015

[101] M E Warkiani A K Tay B L Khoo X Xiaofeng J Han andC T Lim ldquoMalaria detection using inertial microfluidicsrdquo LabChip vol 15 no 4 pp 1101ndash1109 2015

[102] D B Weibel and G M Whitesides ldquoApplications of microflu-idics in chemical biologyrdquoCurrent Opinion in Chemical Biologyvol 10 no 6 pp 584ndash591 2006

[103] G Wu and M H Zaman ldquoLow-cost tools for diagnosing andmonitoring HIV infection in low-resource settingsrdquo Bulletin oftheWorldHealthOrganization vol 90 no 12 pp 914ndash920 2012

[104] L Glendon ldquoHorizon Scan 2015rdquo pp 1ndash43 2015[105] AWMartinez S T Phillips GMWhitesides and E Carrilho

ldquoDiagnostics for the developing world microfluidic paper-based analytical devicesrdquoAnalytical Chemistry vol 82 no 1 pp3ndash10 2010

[106] K Yamada H Shibata K Suzuki and D Citterio ldquoTowardpractical application of paper-based microfluidics for medicaldiagnostics state-of-the-art and challengesrdquo Lab Chip vol 17no 7 pp 1206ndash1249 2017


[107] J Hu S Wang L Wang et al ldquoAdvances in paper-based point-of-care diagnosticsrdquo Biosensors and Bioelectronics vol 54 pp585ndash597 2014

[108] S Liu W Su and X Ding ldquoA Review on Microfluidic Paper-Based Analytical Devices for Glucose Detectionrdquo Sensors vol16 no 12 p 2086 2016

[109] L S A Busa S Mohammadi M Maeki A Ishida H Tani andM Tokeshi ldquoAdvances in microfluidic paper-based analyticaldevices for food and water analysisrdquo Micromachines vol 7 no5 article 86 2016

[110] J KimM Johnson P Hill and B K Gale ldquoMicrofluidic samplepreparation cell lysis and nucleic acid purificationrdquo IntegrativeBiology vol 1 no 10 pp 574ndash586 2009

Submit your manuscripts athttpswwwhindawicom

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PeptidesInternational Journal of


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International Journal of

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GenomicsInternational Journal of


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Microbiology


Table 1 Main characteristics of chemical and mechanical methods to extract nucleic acid (adapted from Harrison 2003)

Method Technique Principle Mode of lysis Cost Most usualapplication References

Chemical

Osmotic shock Osmotic rupture ofmembrane Gentle Cheap Spheroplasts and

Protoplasts [9]

Enzymaticdigestion

Digestion of cellwall Gentle

Cheap at smallscale expensive atlarge scale

Gram-positive andGram-negativebacteria

[13]

Detergents Solubilization ofmembranes Gentle Moderate General use [14]

Alkali treatment Solubilization ofmembrane Harsh Cheap Plasmid DNA [15]

Mechanical

Homogenization(blade or pestle) Shredding of cells Moderate

Moderate (methodof choice for largescale)

Animal tissues [10]

Ultrasonication orcavitation

Disruption of cellsby pressure Harsh Moderate to

expensive

Good forspheroplasts butnot primary cells

[11 12]

Pressure cell(ldquoFrench pressrdquo)

Disruption of cellsby shear force Harsh Moderate

Used forGram-negative andsomeGram-positivebacteria

[6]

Ball millCells crushedbetween glasssteelballsbeads

Harsh Cheap

Used for bacteriayeast microalgaeunicellular animalcells

[15]

of detergents are the main components of chemical lysiswhile mechanical method disrupts the cells by grindingshearing bead beating and shocking [8] It is interestingto note that if one technique does not yield good resultsanothermight prove successful Osmotic shockmethods haveyielded in certain cases better results than common NApurifications protocols such as phenol-chloroform extractionand bead beating [9] Not only is cell disruption importantfor DNA extraction but it also plays a crucial role in thebiopharmaceutical industry as many recombinant proteinsand other important constituents of the cell can be recoveredthrough this process [10ndash12] Another approach for celldisruption is the use of different methods in combinationA good example is the case for enzymatic lysis where manyprotocols use proteases to free the NA from its protectiveprotein scaffold Also the inactivation of cellular nucleasesthat come free into solution in order to protect the newprotein-free NA is crucial [13] A combination of detergentsand chaotropic salts in a single solution is used to solubilizecell wall and or cell membrane and inactivate intracellularnucleases [14 15] Mechanical disruption on the other handmakes use of force to extract out constituents of the cell Aclassic example of grinding in biosciences is the use of mortarand pestle [6] which is nowadays optimized with the useof liquid nitrogen (when allowed by the sample) Cells wallscan also be disrupted by the shock waves created by rapidchanges in pressure elicited by sonication or cavitation [16ndash18] Other mechanical tools available for cell disruption areshearing which use a tangential force to make a hole in the

cell [6] and bead beating which uses different glass or steelbeads to rupture tough cell wall as mentioned by Bunge et al[19] These processes are briefly summarized in Table 1 withconsolidated examples

NAE methods encompass extraction of both DNA andRNA but can be more broadly characterized into chemicallydriven or solid-phase methods both contain the four stepsmentioned above [1 4 5] In the next sections we willreview the working principle of andor rationale for the mainmethods used nowadays in the biological and medical sci-ences Since molecular diagnostics rely heavily on techniquesthat start with NAE we will also discuss some of the basicfeatures of devices available for POC-Dx culminating withthe challenges and limitations of adapting NAE methods topoint-of-care diagnostic tests

2 Chemically Driven Methods

These methods rely on biochemical properties of the cellularcomponents to elicit the desired molecular separation andmight exhibit preference or exclusivity in extracting DNA orRNA depending on its intrinsic characteristics

21 Cesium Chloride (CsCl) Gradient Centrifugation withEthidium Bromide (EtBr) This technique is mainly based onthe phenomenon of buoyant and specific density Ethidiumbromide (EtBr) is an intercalating agent thus reportingthe location of the double-stranded DNA under UV-lightand allowing the easy visual separation of the supercoiled















































[52]




High cost [53]










DNA [66ndash68]






[71 72]

















[49]





High cost [79]


NAE chemicals [80]
























7 Conclusion





Acknowledgments



References

























































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































[52]




High cost [53]










DNA [66ndash68]






[71 72]

















[49]





High cost [79]


NAE chemicals [80]
























7 Conclusion





Acknowledgments



References

























































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






[49]





High cost [79]


NAE chemicals [80]
























7 Conclusion





Acknowledgments



References

























































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















7 Conclusion





Acknowledgments



References

























































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


References

























































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

reviewarticle - oswaldo cruz foundation

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