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DOI:10.1111/eea.12090
TECHNICALNOTE
Aneconomicalandeffectivehigh-throughputDNA
extraction protocol for molecular markeranalysis in
honeybees
BethA.Holloway*,MatthewR.Tarver&ThomasE.Rinderer
USDAHoneyBeeBreeding,Genetics,andPhysiologyLaboratory–MolecularBiology,1157BenHurRoad,BatonRouge,
LA70820,USA
Accepted:7May2013
Key words: Apismellifera,Hymenoptera,purification,nucleicacid
Introduction allow for consistent and reproducible yields, yet can cost
several dollars per sample. Chelating agents purify DNA
The honeybee is becoming an increasingly important with high quality but may require long incubations and
organism in the realm of laboratory-based genetics. As preparations with additional proteinase K (Giraffa et al.,
honey bee health around the world is declining, measures 2000; Casquet et al., 2012). Traditional phenol-chloro-
are being taken to understand the genetic basis of a multi- formextractionsrequirehandling,storage,anddisposalof
tudeoftraitsincludingbehavior,diseaseresistance,surviv- hazardous organic solvents. Honey bee DNA has recently
ability, honey production, and pollination efficiency. beeneffectively extracted and purified by homogenization
Quantitative Trait Loci (QTL) studies and mapping and utilizing proteinase K and ‘salting-out’ methods to
projectsareelucidatingthecomplexinteractionsofgenetic remove proteins (Bourgeois et al., 2008, 2010; Bourgeois
loci that dictate the important traits being bred for by &Rinderer,2009). However,we developed a simple, cost-
beekeepers, queenproducers,andresearchersalike.Quan- effective, and fast method using only sodium chloride and
titative Trait Loci for foraging behavior and aggression sodiumdodecylsulfate(SDS)toextractcleanDNAdirectly
were identified nearly 15 years ago (Hunt et al., 1995, useable for PCR without additional dilution as is typically
1998), long before the advancements of the current and required. This method is particularly useful for high-
ongoing honey bee genome project (Honey Bee Genome throughput extraction of DNA from large numbers of
Sequencing Consortium, Baylor College of Medicine, individual bees while minimizing labor, plastic consum-
Houston,TX,USA).Morerecentstudieshaveidentifieda ables, and reagent requirements. The resulting DNA is of a
single QTL for chalkbrood disease resistance (Holloway highenoughqualityandquantitytodirectlyamplifybands
et al., 2012) andhaveutilizedthegenomedatatofinemap formolecularmarkeranalysis.
the interval to contain just two genes of potential interest
(Holloway et al., 2013). As QTL studies are becoming Materialsandmethods
more commonplace in the understanding of honey bee Beesamples
genetics, DNAextractionmethodsareneedingtobefaster, Freshly emerged adult bees or purple-eyed pupae individu-
moreeffective,andmoreeconomicaltokeeppacewiththe ally pulled from honeycombs were frozen at 20 °C. Bee
analysesofthepopulationsbeingstudied. samplesforagedDNAweremaintainedat20 °Cfor1year
Honey bee QTL studies are generating information on or fresh samples were processed immediately after a lethal
potential gene or allele functionality relevant to mapping freezing. Body segments from fresh or aged samples were
populations. Yet, narrowing the intervals and pinpointing separated and processed with any associated appendages
the genes of interest for eventual marker-assisted selection (headwithantennae,thoraxwithlegsandwings,abdomen).
requires hundreds or even thousands of phenotyped bees
to be processedfor DNAextractionandgenotyping.Typi- Processingmethodology
cally, the extraction processes yield good qualities and 1. Individual bees, pupae, or body segments (or 100 llof
quantities of DNA per individual sample, yet are costly in 1
time and materials. Commercial DNA extraction kits 250 mg ml control BSA) were placed in 96-well
racked 1.2-ml microtiter tubes (Genesee Scientific, San
Diego, CA, USA) containing a single 3.2-mm stainless
*Correspondence:E-mail:beth.holloway@ars.usda.gov steel bead (Next Advanced, Averill Park, NY, USA). A
196 Published2013.ThisarticleisaUSGovernmentworkandisinthepublicdomainintheUSA. EntomologiaExperimentalisetApplicata148:196–200, 2013
HighthroughputDNAextractioninhoneybees 197
second3.2-mmsteelbeadwasloadedintothetubeson squares (LS) means, was used to distinguish differences
top of the tissue and tubes were capped with a silicone amongtreatments.Tukey’sHSDtestwasusedforseparat-
capmat(USAScientific,Ocala,FL,USA). ingmeansbytreatment.
2. Tissues were homogenized by a TissueLyser (Qiagen,
La Jolla, CA, USA) in 285 llof6M NaCl (or in water, Resultsanddiscussion
0.5, 1, 2, 3, 4, or 5 M NaCl) (Fisher Scientific, Pitts-
burgh, PA, USA). Tissue maceration was attained by Increasedsodiumchlorideconcentrationsmoreeffectively‘salt-out’
homogenizing at 30 strokes per s for 2 min; microtiter proteins
plates wererotated,andthehomogenizationrepeated. It is known that the anion products of dissolved salts indi-
3. Plateswerebrieflycentrifuged(BeckmanCoulterAlleg- vidually affect the aggregation or precipitation of proteins
ra X-15 Centrifuge; Beckman Coulter, Brea, CA, USA) fromsolutions such that the efficacies can be described by
to collect the homogenate away from the cap mat. A the Hofmeister series (Zhang & Cremer, 2006; Kunz,
volume of 15 ll of 20% SDS (Amresco, Solon, OH, 2010). Although many DNA extraction protocols (Bour-
USA)wasadded,tubesre-capped,andhomogenizedat geois et al., 2008, 2010; Bourgeois & Rinderer, 2009) and
20strokes per s for 30 s, microtiter plates were rotated, kits use acetate salts (potassium or ammonium), we chose
andthehomogenizationrepeated. to focus on sodium chloride due to its availability and
4. Homogenates were incubated at room temperature for universal use in a variety of laboratories. Chloride anion is
15 min,thencentrifugedat4 800 gfor20 minat4 °C. considered a marginally functional salting-out factor,
5. Aliquots of 150 ll of cleared supernatant were trans- somewhat less effective than acetate on the Hofmeister
ferred to a 96-well PCR plate containing 150 llof series. Typical NaCl concentrations used for fast DNA
20 °Cisopropanol(FisherScientific),andgentlypip- extraction range from less than 1 M (Chen et al., 2010;
ettedupanddownseveraltimes. Margamet al.,2010)toupwardof4 M(Aljanabi&Marti-
6. Plateswerecentrifugedasinstep4topellettheDNA. nez, 1997). We homogenized whole adult bees, whole
7. Supernatant was removed by inverting the plates to pupae, and BSA control protein in a concentration series
decant the liquid and floating gelatinous conglomerate of 0–6 M NaCl (where 0.5 M served as a comparison for
fromthewells. the protocol presented in Margam et al., 2010). During
8. DNApellets were washed twice with 200 llof20 °C this process, we noted an interesting phenomenon follow-
70%ethanol(FisherScientific),followedbycentrifuga- ing the mixing of bee supernatant with isopropanol: the
tions as above in step 4 but modified to 10 min per aggregated protein formed a gelatinous disk at the surface
spin, andsupernatantdecanted. of the supernatant rather than as a pellet at the bottom of
9. DNApellets were dried for 10 min in a 37 °Cincuba- the well, once initial NaCl concentrations reached 5 M.
tor, then resuspendedin25 llpurifiedwater. This resulted in an overall decrease in protein contamina-
tion of the extracted DNA and an easily removable con-
DNAquantificationandqualification glomerate during decanting. The BSA control samples
Resuspended DNA was analyzed by NanoDrop (Nano- fromtheNaClconcentrationserieswereanalyzedforpro-
Drop, Willimgton, DE, USA) for absorbance at k = 260/ tein contamination by measuring absorbance at 280 nm,
1 1
280 nm ratios, dsDNA yield at lg ll , and protein con- where1absorbance is equal to 1 mg ml of protein. The
1
tamination by absorbance 280 = 1mgml .Statistical protein contamination sharply decreases as NaCl concen-
analyseswereperformedusingJMP8.0(Cary,NC,USA). tration increases to 2 M (Figure 1). Bee DNA quantity and
quality measurements following resuspension of the dried
PCRamplification DNA show that the overall DNA yield also decreases
Extracted DNA using 6 M NaCl from frozen samples, somewhat (Figure 2). However, it remains unclear if the
either undiluted or diluted in water (1:20) regardless of 260/280 nmabsorbancemeasurementsusedtodetermine
yield, wasamplifiedbystandardPCRmethods.A155-base extraction efficiency are artificially skewed toward higher
segment of honey bee beta-actin was amplified using DNAyield when protein contamination is high. Regard-
primers F-TGCCAACACTGTCCTTTCTG and R-AGA- less, the quantity and quality are more than sufficient for
ATTGACCCACCAATCCA, then electrophoresed on a downstreamPCRtechniques.
2%agarosegel,andimaged.
ComparablequantitiesandqualitiesofDNAoffreshorhistorical
Statistical analysis tissue samples
All statistical analyses were performed using JMP software Comparisons were made between DNA extractions using
(Version 8). A two-way ANOVA, with adjusted least 6 M NaCl on bees frozen for 1 year and freshly collected
198 Hollowayet al.
A
1
Figure 1 Average( SD;n = 12)proteincontent(mg ml ) B
remainingafterextracting25 mgofBSAusingaconcentration
seriesofNaCl[0.5,1,2,3,4,5,6M,and0(water)].
bees. In addition to whole bees, body segments were
processed to determine whether the yield and quality
are dependent on the type of sample. In a tissue-specific
manner,theextraction protocol yielded comparable DNA
regardless of the age of the sample (Figure 3); however,
only in whole bee or abdomen tissues did the disk form,
float, and easily decant (data not shown). Quantity and
qualityofDNAextractedinthedifferentsamplesweresig-
nificantly different across tissue types (ANOVA; DNA
amount retrieved: whole model, F = 9.53, P<0.0001;
4,95
condition, F = 0.0021, P = 0.96; body region,
1,95 Figure 2 Average( SD;n = 6)quality(solidsymbols)and
F = 12.7, P<0.0001; DNA quality retrieved: whole
3,95 quantity(opensymbols)ofDNAextractedfrom(A)wholebees
model, F = 50.69, P<0.001; condition, F = 0.988,
4,95 1,95 and(B)pupaeusingaNaClconcentrationseries.DNAquality
P = 0.32; body region, F = 67.27, P<0.0001; protein
3,95 remainsrelativelyconstantregardlessoftheNaClconcentration
contamination: whole model, F = 2.78, P = 0.032;
4,85 usedduringtheextraction,whereasoverallDNAyielddecreases
condition, F = 0.11, P = 0.74; body region, F =
1,85 3,85 astheNaClconcentrationincreases.
3.65, P = 0.015. In all cases, n = 12). Despite the remain-
ingextraneousproteinintheheadandthoraxsamples,the
quality and quantity of the extracted DNA is sufficient for wastes for reagents and consumables, while reducing the
subsequentPCRapplicationsonlywhendiluted.Thehead hazards associated with handling organic solvents. This
and thorax DNA samples failed (or partially failed) to protocol essentially uses common, inexpensive, dispos-
directly amplify and required dilution for successful able, and non-hazardous chemicals (NaCl, SDS,isopropa-
amplification (Figure 4A and B), thereby requiring nol, and ethanol). The price of consumables and reagents
additional steps and plastic consumables to make such per 96-well plate of processed DNA is about USD 8.00, or
dilutions. The extraction from abdomens or whole bees 8.5 ¢ per individual bee [with the majority of the price (ca.
resulted in relatively low yield, yet highly purified DNA 80%) determined by the plastic consumables], as com-
such that no dilution step was needed to perform PCR pared to several dollars per extract with commercial kits.
(Figure 4CandD). In addition, sample preparation time is decreased because
wholebeescanbeprocessedasopposedtoindividualbody
Generalremarks segments. The protocol presented here employs a modifi-
We developed a fast, affordable, and eco-friendly DNA cation to other protocols in that the saturated concentra-
extractionprotocolforhighthroughputanalysisofmolec- tionofNaClfunctionstoreduceproteincontaminationto
ular markers in honeybees. The goal of developing this negligible amounts. Importantly, this method allows high-
DNA extraction protocol was to reduce the costs and throughput 96-well-platform extractions as are necessary
HighthroughputDNAextractioninhoneybees 199
A A
B
C
D
B Figure 4 PCRamplificationofa155-bpbeta-actinfragment
usingDNAextractedusing6 MNaClfromindividualtissue
sampleseitherdirectlyfromtheresuspendedpellet(sixlaneson
left) or a 1:20 dilution of the samesamples(sixlanesonright)of
frozen(A)heads,(B)thoraxes,(C)abdomens,or(D)wholebees.
Centerlanesare50-bpladder.Theamplifiedbandsalign
approximatelywiththe150-bpbandoftheladder.
sufficient to perform downstream standard PCR reactions
that enable marker analyses such as cleaved amplified
polymorphisms (Holloway et al., 2013), amplicon
sequencing,andcloningwithhoneybeeDNA.
The quantity of the DNA retrieved is more than suffi-
C cient for standard practices. A return of >5 lgofDNA
fromonetotalindividualbeecanbeexpectedthatismore
than enoughtoperformdozensorhundredsofPCRreac-
tions. Calculations of the total available DNA present in a
whole honeybee suggest that this protocol retrieves only a
portion. Based on the average mass of a nucleotide base-
9
pair (1.029 9 10 pg) (Dolezel et al., 2003), the widely
accepted typical dimensions of a eukaryotic cell (assume
10-lm-diameter sphere, or ca. 0.5 pl), the diploidy of
female bees, and the volume of a newly emerged worker
bee (ca. 100 ll), if the bee were a solid mass of cells (ca.
800 000 cells) then the total mass of DNA contained
would be ca. 92 lg, an extremely gross overestimation
based on the volume of bee that is non-cellularized
Figure 3 Thetimefromsamplecollectiontoprocessingdoesnot (hemolymph) or proteinaceous (exoskeleton) in nature.
affect the (A)overall yield,(B)quality,or(C)purity(protein Regardless, the minimum amount of DNA retrieved is
contamination)oftheextractedDNAafterpurificationwith6 M likely to be at least 5–10% of the total, which is plenty for
NaCl.NostatisticaldifferencesinDNAyield,quality,orpurity standard PCR use. However, because we did not perform
werefoundbetweenfrozen(storedfor1 yearat20 °C)and massspectrometryoranyothermeansofpuritymeasure-
freshlycollectedsamplesforanyofthetissuetypes(head,thorax, ments of the pelleted DNA, we cannot assume that the
abdomen,orwholebody).QuantityandqualityofDNA quantity of DNA calculated by the NanoDrop is entirely
extractedweresignificantlydifferentacrosstissuetypes;different accurateascontaminantsmayalsoaffecttheabsorbanceat
letters onbars withinapanelindicatesignificantdifferences 260 nm used for the determination. Understanding the
(Tukey’sHSDtest:P<0.05). formation of the gelatinous floating disk is beyond the
for a growing array of honey bee genetic studies such as scope of our research, yet the components of it may be
QTLand fine mapping, phylogenetic studies, population sequestering some of the DNA that is lost during the
studies, and the like. The quality of the DNA retrieved is decanting.
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