This document compares four DNA extraction methods for extracting DNA from glacier microorganisms cultivated in the laboratory. The Bosshard-Bano and Zhou methods, which use lysozyme, proteinase K, CTAB and SDS, produced higher DNA yields than the freezing-thawing and Liu methods. Cutting the filter membrane, extracting with chloroform once, and precipitating with ethanol were shown to be more favorable. Comparison to a commercial DNA extraction kit showed that the kit method and optimized Bosshard-Bano method are both effective for studying microbial diversity in glacier surface snow.

1. Methods for extraction of microorganism DNA from glacier surface snow
IntroductionMicroorganismsare aprimarylife forminlow-temperatureenvironments.Theyare
depositedinglaciersthroughatmosphericcirculationandprecipitation(Castello etal.,1999; Sattleret
al.,2001; Priscuet al.,2006). Glaciermicroorganismscaninfluence surface albedoandthe
hydrochemistryof precipitation,andserve asprimaryproducersinmaterial cyclesandenergyflowsof
glaciers(TomasandDuval,1995; Sharp etal.,1999; Tranter etal.,2002; Skidmore etal.,2000, 2005).
The communitystructure anddiversityof culturable microorganismsinice coresandsnow pitshave
beenstudiedbytraditionalculture methods(Christneretal.,2001; Zhang etal.,2003; Miteva etal.,
2004; Xiangetal., 2005; Zhanget al.,2007; Zhanget al.,2007a; Ma etal.,2009).
However,itisestimatedthatonly1%of the microorganismsinenvironmental samplescanbe isolated
(Amannetal.,1995; SchlossandHandelsamn,2003), so accurate environmental microbialstudyis
hampered.
Therefore,since the mid1980s, manymicrobiologistshave investigatedthe amount,community
structure,andfunctionof environmental microorganismsbyculture-independentmethods(Amannet
al.,1995), such as molecularhybridization,electrophoresis(denaturinggradientgel electrophoresis,
DGGE; and temperature gradientgel electrophoresis,TGGE),high-throughputsequencing,andsoon,
but theirusesinthe studyof environmentalmicroorganismsare basedonthe highDNA yieldand
quality.
DNA extractionof environmental microorganismsinvolvessamplepretreatment,cell lysis(physical lysis,
chemical lysis,enzyme lysis,andthe combiningof differentlysispatterns),andextraction,precipitation,
and purificationof DNA.Manybiotechnologycompanieshave producedvariousDNA extractionkitsfor
environmental microorganisms.
Comparedwithtraditional extractionmethods,usingDNA extractionkitsistime- andlabor-saving,
makingthemsuitable forextractingDNA fromscoresof microbial samples.However,DNA extraction
kitsare expensive,sotheirapplicationislimited.Todate,there isnosingle DNA extractionmethod
suitable forall environmental microbial samples.
The biomassof glaciermicroorganismsislow andGram-positive bacteriaare the dominantgroup(Xiang
et al.,2006; Miteva,2008), soit isverydifficulttoestablishahigh-qualityDNA extractionmethod.This
paperreportson certainoptimizedDNA extractionmethodsandcomparesthemwithatypical
commercial kit,andmakesrecommendationsfortheiruse inthe studyof glaciermicroorganisms.
Material and methods:
Bacterial strains:
Gram-positive bacteria(Mycobacteriumspp.,Clavibacterspp.,Pseudonocardiaspp.,andNocardioides
spp.) isolatedfromsnowsamplesandstoredatour laboratorywere culturedwithLBmedium, at25 °C
and 180 rpm, and usedforDNA extraction.

2. Snow samples
In late April andearlyJune 2011, surface snow sampleswere collectedatthe No.12 GlacierinLaohugou
Valley,QilianMountains(39°26′N,96°33′E; 4,600 m a.s.l.) bya technicianwearingcleanclothes,amask,
and PE gloves,andwere placedintosterile bottles.The snow sampleswere transportedtothe
laboratoryandstoredat 15 temperature.
DNA extraction of glaciermicroorganisms:
A total of 1.5 mL of culturedmediumof everybacterial strainwasaddedsuccessivelyintoasingle 2mL
centrifugal tube andcentrifugalizedat8,000 rpm for15 min.Afterwards,the bacterial colonieswere
collectedandtheirDNA wasextractedaccordingtothe followingfourmethods.Everymethodhadthree
repetitions.
DNA extraction according to the Bosshard-Bano method:
We followedthe Bosshard-Banomethodwithone minormodification(Bosshardetal.,2000; Bano and
Hollibaugh,2002).Bacterial colonieswere resuspendedin200 µL solubilizationbuffer(50mMsucrose,
25 mM Tris,10 mM EDTA, pH 8.0, 1.5 M NaCl,1% CTAB),and 50 µL of lysozyme (20mg/mL) incentrifuge
tubes,andthe sampleswere incubatedina37 °C waterbath for1.5 hours.Then10 µL of proteinase K
(20 mg/mL) and15 µL of 20% SDS were added,andthe sampleswere incubatedin37°C and 55 °C water
bathsfor 30 minand 2 hours,respectively.Afterwards,pre-heatedCTAB/NaCl (10% CTABand 0.7 M
NaCl) wasaddedand the sampleswere incubatedina65 °C waterbath for 30 min.
The supernatantswere collectedaftercentrifugationat8,000 rpmfor 15 minand transferredinto2 mL
centrifuge tubes.Supernatantsfromthe extractionswere combinedwithanequal volumeof phenol-
chloroform-isoamylalcohol (25:24:1) and centrifugalizedat12,000 rpm for10 min.The aqueousphase
was recoveredbycentrifugationandprecipitatedwith0.6volume of isopropanol at -20 °C overnight.
The pelletof crude nucleicacidswasobtainedbycentrifugationat12,000 rpm for 20 min,washedtwice
with75% ethanol,anddriedoutat room temperature.The nucleicacidswere resuspendedinTE,giving
a final volume of 20 µL.
DNA extraction according to the Liu method:
Basedon the Liu method(Liuetal.,2009), bacterial colonieswere resuspendedin1mL GTE buffer(25
mM Tris,10 mM EDTA, 50 mMglucose,20 mg/mL, pH 8.0) and the sampleswere incubatedina37 °C
waterbath for2 hours.Then10 µL of proteinase K(20 mg/mL),140 µL of NaCl (5 M), and 55 µL of 20%
SDS were addedandthe mixture wasincubatedat65 °C for 1.5 hours.
Afterextractionwithphenol-chloroform-isoamyl alcohol(25:24:1) and chloroform-isoamyl alcohol
(24:1) and beingcentrifugalizedat10,000 rpmfor 15 min,DNA in the aqueousphase wasprecipitated
withan equal volume of isopropanol at -20 °C overnight.The pelletof crude nucleicacidswasobtained
by centrifugationat12,000 rpm for20 min,washedtwice with75% ethanol,anddriedoutat room
temperature.The nucleicacidswere resuspendedinTE,givingafinal volume of 50 µL.

3. DNA extraction according to the Zhou method:
Basedon the Zhou method(Zhouetal.,1996), bacterial colonieswere resuspendedin650 µL of DNA
extractionbuffer(100mM Tris-HCl (pH8.0), 100 mMsodiumEDTA (pH8.0), 100 mMsodiumphosphate
(pH 8.0), 1.5 MNaCl,1% CTAB),and 5 µL of lysozyme (10mg/mL) incentrifuge tubes,byhorizontal
shakingat 225 rpm for 20 minat 37 °C.Then 5 µL of proteinase K(10 mg/mL) was addedandshakenfor
20 min.Afterwards,75 µL of 20% SDS was added,andthe sampleswere incubatedina65 °C waterbath
for 2 hours withgentle end-over-endinversionsevery15to 20 min.
The supernatantswere collectedaftercentrifugationat8,000 rpm for 15 minand transferredinto2 mL
centrifuge tubes.Supernatantsfromthe extractionswere combinedwithanequal volumeof
chloroform-isoamylalcohol (24:1,vol/vol)andcentrifugalizedat10,000 rpm for 15 min.The aqueous
phase wasprecipitatedwith0.6volume of isopropanol atroomtemperature overnight.The pelletof
crude nucleicacidswasobtainedbycentrifugationat12,000 rpm for 30 min,washedtwice with75%
ethanol,anddriedoutat room temperature.The nucleicacidswere resuspendedinsteriledeionized
water,givingafinal volume of 50 µL.
Freezing-thawing method Bacterial:
colonieswere resuspendedin650 µL of DNA extractionbufferandsubjectedtofreezing-thawingthree
times(Benjaminetal.,2010) andwere thenprocessedaccording tothe Zhou method.
Form of the filter membrane (cut vs. uncut) and optimizing the DNA extraction and
precipitation methods:
1. Afterglaciersurface snowwasmeltedat4 °C (Table 1), about800 mL of meltedwaterwas
averagelyfiltratedintofourpiecesof 0.22 µm filtermembrane.Thentwopiecesof filter
membrane were cutandthe otherswere uncut.Afterwards,eachpieceof filtermembrane was
put ina separate 2 mL centrifuge tube with200 µL of solubilizationbuffer.The subsequent
proceduresof DNA extractionfollowedthe Bosshard-Banomethod.
2. About1,100 mL of meltedwaterwasaveragelyfiltratedintosix piecesof 0.22 µm filter
membrane.Theneachpiece of filtermembrane wasputina separate 2 mL centrifuge tube with
200 µL of solubilizationbuffer.
Three methodswere thenusedforDNA extraction:FL[the firstextractionwithphenol-chloroform-
isoamyl alcohol (25:24:1) andthe secondextractionwithchloroform-isoamylalcohol (24:1)];LII
[extractionwithchloroform-isoamyl alcohol (24:1) twice];andLI [extractionwithchloroform-isoamyl
alcohol (24:1) once].The otherproceduresof DNA extractionfollowedthe Bosshard-Banomethod.
3. About600 mL of meltedwaterwasaveragelyfiltratedintofourpiecesof 0.22 µm filter
membrane.Theneachpiece of filtermembrane wasputina separate 2 mL centrifuge tube with
200 µL of solubilizationbuffer.Twodepositionmethodswere usedforDNA precipitation:Y
(depositionwiththree volumesof pure ethanol),andB(depositionwith2/3volume of
isopropanol).

4. The otherproceduresof DNA extractionfollowedthe Bosshard-Banomethod.Eachof these
treatmentswasduplicated,andthe DNA yieldof the firstwaterbathandthe mixture DNA yieldof the
secondandthird waterbathwere calculated.
Comparing the optimizedextractionmethods withkit extraction :
The optimizedBosshard-Banomethodandthe Zhoumethodwere namedZEandZH, respectively.About
600 mL of meltedwaterwasaveragelyfiltratedintofourpiecesof 0.22 µm filtermembrane.ThenDNA
was extractedwithaMo-Biokit(PowerWaterDNA IsolationKit,No.14900-50-NF,Mo-BioLaboratories,
Carlsbad,CA,USA),the ZE and ZH methodswere applied,andthe bettermethodof glacier
microorganismDNA extractionwasdetermined.
The quantity of microbial DNA :
The DNA concentrationandoptical densityvaluesat230, 260, and 280 nm were estimatedwithanND-
1000 spectrophotometer(NanoDropProducts,Wilmington,DE,USA),andDNA puritywas assessedwith
A260/A230 (DNA/humicacid) andA260/A280 (DNA/protein).
PCR amplification:
DNA extractedwiththe Mo-Biokitandthe ZE andZH methodswere amplifiedbyPCRusingthe
oligonucleotide primers8-27f (5’-AGAGTTTGATCCTGGCTCAG-3’) and1507-1492r (5’-
CGGTTACCTTGTTACGACTT-3’) (Whitakeretal.,2003).
PCR was carriedoutin a final volume of 25 µL using4-µL template DNA,2.5mM MgCl2, 0.2 mMdNTP,
0.2 µM eachprimer,and1 U Taq polymerase (MBI).Topreventcontamination,anegativeparallel
amplificationwasestablishedbyautoclaveddeionizedwater.Reactionswere performedwiththe
followingcyclingparameters:94 °C for1 minfor an initial denaturation,followedby30 cyclesof 94 °C

5. for 1 min,58 °C for 1 min,and72 °C for1.5 min,and a final incubationat72 °C for10 min.The products
of PCRamplificationwere estimatedwith1.0% agarose gel electrophoresis.
Resultsand discussion:
Comparisonof differentDNA extractionmethodsThe A260/A280 ratiosof the fourmethodsranged
from1.8 to 2.0 (Table 2),whichindicatedthatthe extractingeffectsof phenol andchloroformwere
good;theirA260/A230 ratioswere >2 (Manchester,1995; Small et al.,2001), whichindicatedthatthere
were fewresidualsof reagentsandimpurities.
The discrepancyof the DNA yieldswasbasicallyattributable tothe differentlysispatterns,because our
experimental sampleswere mixturesof pure cultures,notenvironmental samplescontaininghumicacid
and so onGram-positive bacteriaare the dominantgroupof glacierbacteria.
Theyadapt to extreme low-temperature environmentswiththe complex structure of theircell
walls(Miteva,2008), sowe testedfourmethodswithdifferentlysispatternsforextractingDNA
fromthe mixture of Gram-positive bacteria.Inlysispatterns,lysozyme andproteinKcandissolve
cell wallsandprotein,respectively.CTABandSDScan decompose the lipidbilayer,andrepeated
freezing-thawingcanbreakdownthe cell structure.
ConclusionFourDNA extractionmethodswithdifferentlysispatternswereusedforextractingthe
DNA of glaciermicroorganisms(Gram-positivebacteria) cultivatedinourlaboratory,andthe DNA
yieldsof these fourmethodswereestimated.The lysispatternsof the Bosshard-BanoandZhou
methods(i.e.,lysozyme-proteinK-CTAB-SDS) were betterthanthose of the freezing-thawingand
the Liu methods.
Procedurally,cuttingthe filtermembrane,extractingwithchloroformonlyonce,andprecipitating
withethanol were showntobe more favorable forDNA extractionof glaciermicroorganisms.
Comparisonof these optimizedtraditionalextractionmethodswiththe Mo-Biokitshowedthat,
basedon DNA parametersandPCR amplification,the kitmethodissuitableforstudyof microbial
diversityinglaciersurface snow,whilethe Bosshard-Banomethodisalsoaneffective andless
expensive glaciermicroorganismDNA extractionmethod.

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