Field preservation and optimization of a DNA ... - Porifera Brasil

Porifera Research: Biodiversity, Innovation and Sustainability - 2007555Field preservation and optimization of a DNAextraction method for PoriferaAdriana Salgado (1) , Thomáz Vieiralves (1) , Flávia R.M. Lamarão (1) , Leonardo L.M. Assumpção (1) , DéboraGomes (2) , Lia Jascone (2) , Ana Luiza Valadão (2) , Rodolpho M. Albano (3) , Gisele Lôbo-Hajdu (1*)(1)Departamento de Biologia Celular e Genética/DBCG, Instituto de Biologia Roberto Alcantara Gomes/IBRAG,Universidade do Estado do Rio de Janeiro/ UERJ, Rua São Francisco Xavier, 524 – PHLC, sala 205, Maracanã, 20550-013, Rio de Janeiro, RJ, Brazil(2)Curso de Ciências Biológicas, Disciplina de Genética Básica, DBCG, IBRAG, UERJ(3)Departamento de Bioquímica/DBq, IBRAG, UERJ, Av. 28 de Setembro, 87 fundos, PAPC 4o andar, 20551-013,Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. lobohadju@oi.com.brAbstract: The small number of molecular studies on lower invertebrates may be due to a limited availability of fresh orproperly preserved biological material. Specimens collected and preserved in different fixatives can influence the quality ofthe extracted DNA. Variables such as the type of fixative, time of storage, and extraction protocol are critical for obtainingDNA in sufficient quantity and of good quality. This work evaluates the efficiency of six different field fixatives and the mosteffective DNA extraction protocol for marine sponges. Sponges were collected and preserved in one of the following: 1) 96%ethanol, 2) 70% ethanol, 3) dry-ice, 4) air-dried, 5) lyses buffer with guanidine hydrochloride (LBWGH), or 6) silica gel.Genomic DNA was extracted by one of four different protocols: lyses buffer with proteinase K, cetyl trimethyl ammoniumbromide (CTAB), guanidine hydrochloride or DNAzol ® . The quality of the DNA obtained was determined with scores ofthe DNA degradation level. Our results showed that high molecular weight DNA was seen with all six fixatives albeit with agreat variation in DNA quality. Based on gel analysis, the most effective preservation methods, both in quality and quantity,were dry ice, silica gel and LBWGH. Regarding the DNA extraction procedures, CTAB, LBWGH and the DNAzol ® methodsproduced high quality genomic DNA. However, considering the cost-benefit ratio of the methods for the processing of a largenumber of samples, a short term preservation in combination with extraction with LBWGH is the best protocol among thetechniques tested here.Keywords: field preservation, DNA extraction, method optimization, PoriferaIntroductionThe constant need to unravel the phylogenetic relationshipsof various organisms and the popularization of molecularmethods transformed museum collections in valuable sourcesof DNA. Biologists have been extracting DNA from specimensdeposited in collections for decades and the protocols to usethis material have been continuously improving (Arrighi etal. 1968, Pääbo 1989, Post et al. 1993, Thomas 1994, Reiss etal. 1995, Dilon et al. 1996, Hammond et al. 1996, Shedlocket al. 1997, Kalmár et al. 2000, Berntson and France 2001,Rohland et al. 2004, Chakraborty et al. 2006). However, theintegrity of the extracted DNA will vary according to theorganism, preservation conditions, time of storage and DNAextraction method.Few reports comparing preservation methods have beenpublished for marine invertebrates (France and Kocher1996, Chase et al. 1998, Dawson et al. 1998, Berntson andFrance 2001, Crabbe 2003), and, very recently, one studyhas addressed members of Porifera (Ferrara et al. 2006).Overcoming the preservation step, the next decision is tochoose a reliable DNA extraction method. However, asobserved for several other marine invertebrates, the qualityof the DNA extracted from sponges is rarely suitable for PCRreactions. One possible explanation can be the presence ofacidic polysaccharides in several marine sponges, whichcould inhibit PCR amplification (Demeke and Adams 1992).Furthermore, it was previously reported that the yield ofDNA extraction varies considerably among taxa and shouldnot be extrapolated from one organism to another (Dillon etal. 1996, Dawson et al. 1998, Mtambo et al. 2006).Several nucleic acid extraction procedures are currentlyavailable. They go from a a simple salting out method to theuse of complex buffers with high salt, detergents and reagentswhich cleave disulfide bridges (Miller et al. 1988, Boom etal. 1990, Seutin et al. 1991, Hong et al. 1997, Dawson etal. 1998, Berntson and France 2001, Mtambo et al. 2006,Ferrara et al. 2006). The aim of this work was to assess simpleprotocols for the preservation and DNA extraction frommarine sponges. For this, different protocols were comparedto evaluate the quality and quantity of the extracted genomicDNA and its suitability for PCR amplification.

557Fig. 2: DNA quality state determination and scores from samplesof Hymeniacidon heliophila preserved in six different fixatives andextracted by the LBWGH method. Concentration markers are 10 ngand 50 ng lambda DNA. D = air dried, E70 = 70% ethanol, E96 =96% ethanol, F = frozen in dry-ice, L = LBWGH solution and S =silica gel.Fig. 3: Agarose gel (2%) electrophoresis showing ITS-1 PCRamplification from one H. heliophila individual preserved in sixdifferent fixatives. M = 100bp ladder molecular weight marker. D= air dried, E70 = 70% ethanol, E96 = 96% ethanol, F = frozen indry-ice, L = LBWGH solution and S = silica gel.of 5. Despite the variation in DNA quantity and quality, allextraction procedures produced DNA that rendered singlePCR products for both ITS-1 and ITS-2. Figure 3 shows the380 bp fragment resultant of ITS-1 PCR amplification fromH. heliophila.To test long-term preservation, each sample, sorted by thefixation procedures used was submitted to the LBWGH DNAextraction method after seven, 15, 30 and 60 days of storage.The quality of the DNA extracted from the seven and 15 dayssamples is shown in Fig. 4. It can be clearly seen that after aweek all yields decrease, which was reflected on the qualityscores. Nevertheless, even after 60 days of storage at roomtemperature, enough DNA to be amplified could be extracted(data not shown).The best quality DNA was obtained preserving sponges indry-ice followed by freezing at -80 o C. LBWGH and silica gelwere also good fixatives. The other tested fixatives yieldedDNA with less quality according to the following order, frombest to worst: air-dried, 96% ethanol and 70% ethanol for H.heliophila.Other tested sponges gave different results. For example, A.viridis showed excellent quality DNA for air-dried preservedsamples. One possible justification for these discrepancies isthe different composition of sponge tissues and the amountof polysaccharides they possess. Amphimedon viridis can besqueezed until almost all water is removed, which facilitatesthe denaturing of nucleases (DNase and RNAse). So, for A.viridis, samples preserved in a dry state, yielded DNA as goodas those kept frozen or in LBWGH solution.Two other sponges tested, P. magna and A. fulva, werewell preserved in LBWGH, frozen and in 96% ethanol. Theydiffer from A. viridis and H. heliophila regarding the useof 70% ethanol, silica gel and air for fixation. Paraleucillamagna would allow high quality DNA extractions from thesethree fixatives which imply in quick removal of water, whileA. fulva provided low quality, if any, genomic DNA. Again,these two sponges presented different consistencies, beingP. magna more friable and A. fulva more meaty (firm whenpressed).These results demonstrated that LBWGH solution yieldedreasonable DNA, both in quality and in quantity. This solutionacts denaturing nucleases because it contains a chaotropicagent (guanidine hydrochloride), EDTA, which absorbsCa ++ and Mg ++ essential ions for some nucleases, and N’-laurylsarcosine, a detergent responsible by the disintegrationof cellular membranes, allowing the release of nuclear DNA.Although the LBWGH solution produced better results than96% ethanol, it did not preserve the shape and integrity of theorganism. Guanidine hydrochloride cannot be considered asa permanent fixative for biological material, but it is perfectlyviable as a temporary transport solution of specimens forDNA assays from the field to the laboratory. If kept frozen(-20 o C), samples in LBWGH can render DNA suitable forPCR amplification up to 10 years after being collected (datanot shown).Additional problems related to shipping preservedbiological specimens were introduced by new rules imposedby the International Air Transport Association (IATA). Ina query launched at the Porifera Mailbase (see Archives ofPorifera at http://www.jiscmail.ac.uk/lists/porifera.html) byDr John Hooper, Queensland Museum, on January 24 th 2005,a consensus list of recommended preservative methods forsponges was announced. Three methods for preserving andshipping overseas sponge samples for DNA studies were: 1)small fragments mummified in high analytical grade silicagel, 2) samples immersed in DMSO buffer (20% DMSO, 250mM EDTA, NaCl to saturation, pH 8.0) (adapted from Seutinet al. 1991), and 3) freeze-dried sponges.More recently, the subscribers of the Porifera Mailbase oncemore inquired about sponge preservation for genetic work (Dr.Claire Goodwin, Ulster Museum, on May 25 th 2006). Fromthat debate additional suggestions were made: preservingsmall pieces in RNALater ® (Ambion), 95% ethanol, 70%ethanol (1:10 weight sponge to ethanol volume), isopropanol,

558Fig. 4: DNA quality statedetermination and scores fromsamples of H. heliophila preservedin six different fixatives for sevenand fifteen days. Concentrationmarkers are 10, 20, 50 and 100 ng,respectively). D = air dried, E70 =70% ethanol, E96 = 96% ethanol,F = frozen in dry-ice, L = LBWGHsolution and S = silica gel.lyses buffer with high concentration of a chaotropic agent,such as guanidine hydrochloride (GuHCl), and FTA ® ClassicCards (Whatman BioScience, Fast Technology for Analysisof nucleic acids) (Crabbe 2003).Considering altogether, for small to medium projects, itis viable to collect samples of sponges in LBWGH solution.Large marine faunistic surveys will demand a less expensiveand more easily accessible fixative. In that case, 96% ethanolis recommended to preserve Porifera samples, which keepthe morphology of the sample while yielding an acceptableamount of good quality DNA.One point to be highlighted is the importance of taking onlya small sample when preserving in silica gel and LBWGH.The same probably stands true for some species preserved in96% or 70% ethanol (Seutin et al. 1991, Reiss et al. 1995).Dessauer et al. (1996) suggested that samples should be cutin fragments not bigger than 1 mm 3 , although Dawson et al.(1998) had success with 0.2 cm 3 finely chopped tissues. Thisfeature is important because the essential step in alcohol andsilica gel preservation is the fast elimination of water out ofthe tissues in order to limit hydrolytic cleavage of the nucleicacids.Thus, the sponge should be cleaned and the sea waterdrained just after collection and separated in two subsamples.A bigger piece, with visible representative anatomicfeatures, should be placed in 96% ethanol. Even drained, thesponge will keep some sea water inside that will lower theethanol concentration. After some days, the ethanol shouldbe replaced. A second, smaller piece from the clean inner partof the sponge should be cut in small fragments and placed inLBWGH, silica gel, RNALater ® or FTA ® Cards.A comparison between the four extraction methodsapplied to fresh material is shown in Fig. 5. Notably,LBWGH and LBWPK extraction methods if combined(Fig. 5, LPK) yield genomic DNA with improved quality.DNAzol ® rendered genomic DNA with higher quality scorethan the other techniques. This kit, and others such as Trizol ®and RNALater ® , has guanidine hydrochloride (GuHCl) orthiocyanate (GuSCN) on its composition (Boom et al. 1990)and therefore resembles the LBWGH method.The CTAB buffer works well for fresh (Fig. 5, CT) andethanol preserved samples, although resulted in an smalleramount of DNA. The combination of CTAB with proteinaseK reduces so much the amount of DNA obtained that it is notvisible in agarose gels (Fig. 5, CTK).Finally, in Fig. 6, a comparison of preservation andextraction methods is shown. Sponge samples preserved andextracted in/with LBWGH buffer resulted in perfect DNA forPCR amplification.A proposal taken from this work is the use of the LBWGHbuffer for the short-time fixation of sponge samples at roomtemperature, followed by the LBWGH plus proteinase KDNA extraction. Being a simple solution, like RNALater®,the LBWGH should not be a problem to exchange loans byconventional mail. The use of screw capped 2 mL vials isrecommended, always using three times more LBWGH bufferthan sponge fragments. For long-time fixation in LBWGHbuffer, samples should be kept at -20 o .C.AppendixMethod 1, Lyses Buffer with Proteinase K (LBWPK):Homogenization in 1:3 (weight:volume) solution of SETbuffer (0.15 M NaCl, 0.05 M Tris/HCl pH 8.0, 10 mM EDTA,0.4% SDS) plus 20 µg/µL proteinase K. The suspension wasincubated at 55ºC for 1 hr and centrifuged at 3000 g/10 min.The supernatant was extracted once with an equal volumeof phenol:chloroform:isoamyl alcohol (25:24:1) and twicewith one volume of chloroform. Precipitate DNA fromthe homogenate by the addition of 2 volumes of ethanolplus 1/10 volume of 3 M sodium acetate pH 5.2, followedby centrifugation at 10000 g/10 min at 4ºC. The pellet waswashed in 70% ethanol, air dried, dissolved in sterile waterplus 20 μg/ml RNAse A (GIBCO BRL), and incubated for 1h at 37ºC.

559Fig. 5: DNA quantification andscores from fresh collected samplesshowing the extraction productsfrom two different individuals ofH. heliophila with four differentmethods and combinations of them.Concentration markers are 10,20, 50 and 100 ng, respectively).L = LBWGH solution, LPK =LBWGH plus proteinase K, Dz= DNAzol, CT = CTAB solution,CK = CTAB plus proteinase K, PK= proteinase K.Method 2, Lyses Buffer with Cetyl Trimethyl AmmoniumBromide (CTAB): Homogenization of sponge fragmentsin 1:3 (weight:volume) solution of 2% CTAB in 100 mMTris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl plus 1 µL β-mercaptoetanol and 10 µg/µL proteinase K. The suspensionwas incubated at 50ºC for at least 1 hr and centrifuged at3000 g/10 min. The supernatant was extracted twice with onevolume of chloroform: isoamyl alcohol (24:1). PrecipitateDNA from the homogenate by the addition of 0.8 volumesof isopropanol plus 1/10 volume of 3 M sodium acetate pH5.2, followed by centrifugation at 10000 g/10 min at 4ºC.The pellet was washed in 70% ethanol, air dried, dissolvedin sterile water plus 20 μg/ml RNAse A (GIBCO BRL), andincubated for 1 h at 37ºC.Method 3, Lyses Buffer with Guanidine Hydrochloride(LBWGH): Specimens were ground with a rod in a mortar with1:5 (weight:volume) solution of 4 M guanidine hidrochloride,50 mM Tris-HCl pH 8.0, 0.05 M EDTA, 0.5% sodium-N’-lauroylsarcosine and 1% ß-mercaptoethanol. The suspensionwas incubated at 50ºC for 1 hr and centrifuged at 3000 g/10min. The supernatant was extracted with an equal volumeof phenol:chloroform:isoamyl alcohol (25:24:1) and nucleicacids were precipitated with 2 volumes of ethanol. The pelletwas washed in 70% ethanol and air dried. The dried pellet wasdissolved in sterile water plus 20 μg/ml RNAse A (GIBCOBRL) and incubated at 37ºC for 2 h.Method 4, DNAzol®: Homogenization of 25-50 mg fragmentsin 1 ml of DNAzol® in a hand held homogenizer by applyingas few strokes as possible. The homogenate was centrifugedat 3000 g/10 min. The supernatant was transferred to a freshtube and the DNA precipitated by the addition of 0.5 ml of100% ethanol per 1 ml of DNAzol® used for the isolation.The DNA precipitate was removed by spooling with a pipettetip or by centrifugation at 10000 g/10 min at 4ºC. DNA pelletwas washed twice with 75% ethanol and dissolved in water.Fig. 6: DNA quantification of total genomic DNA extracted by theLBWGH method. Molecular weight marker are from top to bottom:23100, 9400, 6600, 4400, 2300, 2000 bp. E96 = sponge specimenspreserved at museum collections for long term in ethanol 96%, andL = sponge specimens preserved directly in LBWGH.AcknowledgementsD. M. Braga de Mello is thanked for technical assistance. This workwas carried out with financial assistance from Programa Prociência,Sub-reitoria de Pós-graduação e Pesquisa (SR2-UERJ), FundaçãoCarlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de

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