Porifera Research: Biodiversity, Innovation and Sustainability - 2007
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Field preservation and optimization of a DNA extraction method for Porifera Adriana Salgado(1), Thomáz Vieiralves(1), Flávia R.M. Lamarão(1), Leonardo L.M. Assumpção(1), Débora Gomes(2), Lia Jascone(2), Ana Luiza Valadão(2), Rodolpho M. Albano(3), Gisele Lôbo-Hajdu(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, 20550013, 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.
[email protected] (1)
Abstract: The small number of molecular studies on lower invertebrates may be due to a limited availability of fresh or properly preserved biological material. Specimens collected and preserved in different fixatives can influence the quality of the extracted DNA. Variables such as the type of fixative, time of storage, and extraction protocol are critical for obtaining DNA in sufficient quantity and of good quality. This work evaluates the efficiency of six different field fixatives and the most effective 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 ammonium bromide (CTAB), guanidine hydrochloride or DNAzol®. The quality of the DNA obtained was determined with scores of the DNA degradation level. Our results showed that high molecular weight DNA was seen with all six fixatives albeit with a great 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® methods produced high quality genomic DNA. However, considering the cost-benefit ratio of the methods for the processing of a large number of samples, a short term preservation in combination with extraction with LBWGH is the best protocol among the techniques tested here. Keywords: field preservation, DNA extraction, method optimization, Porifera
Introduction The constant need to unravel the phylogenetic relationships of various organisms and the popularization of molecular methods transformed museum collections in valuable sources of DNA. Biologists have been extracting DNA from specimens deposited in collections for decades and the protocols to use this material have been continuously improving (Arrighi et al. 1968, Pääbo 1989, Post et al. 1993, Thomas 1994, Reiss et al. 1995, Dilon et al. 1996, Hammond et al. 1996, Shedlock et al. 1997, Kalmár et al. 2000, Berntson and France 2001, Rohland et al. 2004, Chakraborty et al. 2006). However, the integrity of the extracted DNA will vary according to the organism, preservation conditions, time of storage and DNA extraction method. Few reports comparing preservation methods have been published for marine invertebrates (France and Kocher 1996, Chase et al. 1998, Dawson et al. 1998, Berntson and France 2001, Crabbe 2003), and, very recently, one study has addressed members of Porifera (Ferrara et al. 2006). Overcoming the preservation step, the next decision is to choose a reliable DNA extraction method. However, as
observed for several other marine invertebrates, the quality of the DNA extracted from sponges is rarely suitable for PCR reactions. One possible explanation can be the presence of acidic polysaccharides in several marine sponges, which could inhibit PCR amplification (Demeke and Adams 1992). Furthermore, it was previously reported that the yield of DNA extraction varies considerably among taxa and should not be extrapolated from one organism to another (Dillon et al. 1996, Dawson et al. 1998, Mtambo et al. 2006). Several nucleic acid extraction procedures are currently available. They go from a a simple salting out method to the use of complex buffers with high salt, detergents and reagents which cleave disulfide bridges (Miller et al. 1988, Boom et al. 1990, Seutin et al. 1991, Hong et al. 1997, Dawson et al. 1998, Berntson and France 2001, Mtambo et al. 2006, Ferrara et al. 2006). The aim of this work was to assess simple protocols for the preservation and DNA extraction from marine sponges. For this, different protocols were compared to evaluate the quality and quantity of the extracted genomic DNA and its suitability for PCR amplification.
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Material and methods Collection, preservation and storage Specimens of Hymeniacidon heliophila Parker, 1910 and Paraleucilla magna Klautau, Monteiro and Borojevic, 2004 (Calcarea) were collected at Praia Vermelha beach, Rio de Janeiro (23°00’S, 43°12’W), while Amphimedon viridis Duchassaing and Michelloti, 1864 and Aplysina fulva (Nardo, 1834) were collected at João Fernandinho beach, Búzios, Rio de Janeiro (22º44’S, 41º54’W). After careful dissection to remove macroscopic symbionts and substrate debris, all individuals were divided in six fragments, each one preserved differently: 1) 96% ethanol, 2) 70% ethanol, 3) frozen in dryice (solid CO2), 4) air-dried, 5) lyses buffer with guanidine hydrochloride (LBWGH - 4 M guanidine hydrochloride, 50 mM Tris-HCl pH 8.0, 0.05 M EDTA, 0.5% sodium-N’lauroylsarcosine, 1% ß-mercaptoethanol), and 6) silica gel. Three replicates of all samples were preserved in each fixative for seven to sixty days at room temperature. The exception were the air-dried samples, which were kept after one day in the oven at 60oC and the fragments frozen in dryice, which were stored in -80oC ultra freezer. In order to compare the preservation protocols, DNA was extracted from these samples by the same method (LBWGH, see below).
DNA Extraction Hymeniacidon heliophila was collected, divided in four fragments of same size and wet weight, and frozen in dry-ice immediately after removal from sea water. Afterwards, the samples were submitted to four methods of genomic DNA extraction (described in the appendix): - Method 1, Lyses buffer with proteinase K (LBWPK); - Method 2, Lyses buffer with cetyl trimethyl ammonium bromide (CTAB); - Method 3, Lyses buffer with guanidine hydrochloride (LBWGH); - Method 4, DNAzol.
Determination of quality and quantity of DNA The DNA concentration was estimated in 0.8% agarose gels run in TBE 0.5X (50 mM Tris Base, 50 mM boric acid, 1 mM EDTA) by comparison with solutions of known concentrations of lambda bacteriophage genomic DNA (10 ng/µl, 20 ng/µl, 50 ng/µl, 100 ng/µl), and visualized under UV light after staining with 0.6 µg/ml ethidium bromide. To standardize the quantification, each sample applied on a gel had its volume adjusted according to the initial wet weight. In order to evaluate the quality of the genomic DNA, the scores given in Amos and Hoelzel (1991) were adopted, with some modifications. For each sample, a score from 0 to 5 was given, depending on the degree of DNA degradation. A diagrammatic representation of how those scores were correlated to DNA degradation when visualized in agarose gels is presented in Fig. 1.
Fig. 1: Degradation scores for analysis of DNA quality, modified from Amos and Hoelzel (1991). 1 = not degraded high weight DNA, 2 = high weight DNA with little degradation, 3 = high weight DNA with degradation, 4 = DNA with high degradation, 5 = completely degraded DNA, and 6 = no DNA.
PCR Amplification test Two pairs of primers (Lobo-Hajdu et al. 2004): 18S Forward /5‘-TCATTTAGAGGAAGTAAAAGTCG-3‘, 5,8S Reverse /5‘-GCGTTCAAAGACTCGATGATTC-3‘, and 5,8S Forward /5‘-GAATCATCGAGTCTTTGAGC C-3‘, 28S Reverse /5‘-GTTAGTT TCTTTTCCTCCGCTT-3‘ were used to test the amplification of the two internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal RNA (rRNA). Each 30 µl PCR amplification reaction mixture contained 10 ng of genomic DNA, reaction buffer (10 mM KCl, 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 0.1% Triton-X-100, 100 mg/ml gelatin), 3 mM MgSO4, 200 µM dNTPs, 80 ng of each primer and 1 unit of DNA polymerase (Platinum Taq, Invitrogen, SP, Brazil). PCR amplification was carried out in a DNA thermal cycler (M.J. Research PTC-100). An initial denaturation step of 5 min at 96ºC was followed by 35 cycles of 30 s at 94ºC, 45 s at 52ºC and 1 min at 72ºC, with an additional final step of 5 min at 72ºC for final expansion. The amplified bands were separated by electrophoresis on 2% agarose gels in 0.5X TBE. The size of the amplified fragments was estimated by comparison with standard DNA ladders.
Results and discussion All of the six fixation procedures tested allowed for some extracted DNA. However, depending on the fixative used a great variation on DNA amount and quality was observed (Fig. 2). LBWGH, freezing and silica gel dried samples obtained the best scores (scores 1 and 2). Even though air-dried samples resulted in good quality DNA (score 3), the yield was lower than with the above mentioned methods. The worst quality results were obtained for 70% ethanol with a score
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Fig. 2: DNA quality state determination and scores from samples of Hymeniacidon heliophila preserved in six different fixatives and extracted by the LBWGH method. Concentration markers are 10 ng and 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 PCR amplification from one H. heliophila individual preserved in six different fixatives. M = 100bp ladder molecular weight marker. D = air dried, E70 = 70% ethanol, E96 = 96% ethanol, F = frozen in dry-ice, L = LBWGH solution and S = silica gel.
of 5. Despite the variation in DNA quantity and quality, all extraction procedures produced DNA that rendered single PCR products for both ITS-1 and ITS-2. Figure 3 shows the 380 bp fragment resultant of ITS-1 PCR amplification from H. heliophila. To test long-term preservation, each sample, sorted by the fixation procedures used was submitted to the LBWGH DNA extraction method after seven, 15, 30 and 60 days of storage. The quality of the DNA extracted from the seven and 15 days samples is shown in Fig. 4. It can be clearly seen that after a week all yields decrease, which was reflected on the quality scores. Nevertheless, even after 60 days of storage at room temperature, enough DNA to be amplified could be extracted (data not shown). The best quality DNA was obtained preserving sponges in dry-ice followed by freezing at -80oC. LBWGH and silica gel were also good fixatives. The other tested fixatives yielded DNA with less quality according to the following order, from best 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 preserved samples. One possible justification for these discrepancies is the different composition of sponge tissues and the amount of polysaccharides they possess. Amphimedon viridis can be squeezed until almost all water is removed, which facilitates the denaturing of nucleases (DNase and RNAse). So, for A. viridis, samples preserved in a dry state, yielded DNA as good as those kept frozen or in LBWGH solution. Two other sponges tested, P. magna and A. fulva, were well preserved in LBWGH, frozen and in 96% ethanol. They differ from A. viridis and H. heliophila regarding the use of 70% ethanol, silica gel and air for fixation. Paraleucilla magna would allow high quality DNA extractions from these three fixatives which imply in quick removal of water, while A. fulva provided low quality, if any, genomic DNA. Again, these two sponges presented different consistencies, being
P. magna more friable and A. fulva more meaty (firm when pressed). These results demonstrated that LBWGH solution yielded reasonable DNA, both in quality and in quantity. This solution acts denaturing nucleases because it contains a chaotropic agent (guanidine hydrochloride), EDTA, which absorbs Ca++ and Mg++ essential ions for some nucleases, and N’laurylsarcosine, a detergent responsible by the disintegration of cellular membranes, allowing the release of nuclear DNA. Although the LBWGH solution produced better results than 96% ethanol, it did not preserve the shape and integrity of the organism. Guanidine hydrochloride cannot be considered as a permanent fixative for biological material, but it is perfectly viable as a temporary transport solution of specimens for DNA assays from the field to the laboratory. If kept frozen (-20o C), samples in LBWGH can render DNA suitable for PCR amplification up to 10 years after being collected (data not shown). Additional problems related to shipping preserved biological specimens were introduced by new rules imposed by the International Air Transport Association (IATA). In a query launched at the Porifera Mailbase (see Archives of Porifera at http://www.jiscmail.ac.uk/lists/porifera.html) by Dr John Hooper, Queensland Museum, on January 24th 2005, a consensus list of recommended preservative methods for sponges was announced. Three methods for preserving and shipping overseas sponge samples for DNA studies were: 1) small fragments mummified in high analytical grade silica gel, 2) samples immersed in DMSO buffer (20% DMSO, 250 mM EDTA, NaCl to saturation, pH 8.0) (adapted from Seutin et al. 1991), and 3) freeze-dried sponges. More recently, the subscribers of the Porifera Mailbase once more inquired about sponge preservation for genetic work (Dr. Claire Goodwin, Ulster Museum, on May 25th 2006). From that debate additional suggestions were made: preserving small pieces in RNALater® (Ambion), 95% ethanol, 70% ethanol (1:10 weight sponge to ethanol volume), isopropanol,
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Fig. 4: DNA quality state determination and scores from samples of H. heliophila preserved in six different fixatives for seven and fifteen days. Concentration markers are 10, 20, 50 and 100 ng, respectively). D = air dried, E70 = 70% ethanol, E96 = 96% ethanol, F = frozen in dry-ice, L = LBWGH solution and S = silica gel.
lyses buffer with high concentration of a chaotropic agent, such as guanidine hydrochloride (GuHCl), and FTA® Classic Cards (Whatman BioScience, Fast Technology for Analysis of nucleic acids) (Crabbe 2003). Considering altogether, for small to medium projects, it is viable to collect samples of sponges in LBWGH solution. Large marine faunistic surveys will demand a less expensive and more easily accessible fixative. In that case, 96% ethanol is recommended to preserve Porifera samples, which keep the morphology of the sample while yielding an acceptable amount of good quality DNA. One point to be highlighted is the importance of taking only a small sample when preserving in silica gel and LBWGH. The same probably stands true for some species preserved in 96% or 70% ethanol (Seutin et al. 1991, Reiss et al. 1995). Dessauer et al. (1996) suggested that samples should be cut in fragments not bigger than 1 mm3, although Dawson et al. (1998) had success with 0.2 cm3 finely chopped tissues. This feature is important because the essential step in alcohol and silica gel preservation is the fast elimination of water out of the tissues in order to limit hydrolytic cleavage of the nucleic acids. Thus, the sponge should be cleaned and the sea water drained just after collection and separated in two subsamples. A bigger piece, with visible representative anatomic features, should be placed in 96% ethanol. Even drained, the sponge will keep some sea water inside that will lower the ethanol concentration. After some days, the ethanol should be replaced. A second, smaller piece from the clean inner part of the sponge should be cut in small fragments and placed in LBWGH, silica gel, RNALater® or FTA® Cards. A comparison between the four extraction methods applied 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 score than the other techniques. This kit, and others such as Trizol® and RNALater®, has guanidine hydrochloride (GuHCl) or
thiocyanate (GuSCN) on its composition (Boom et al. 1990) and therefore resembles the LBWGH method. The CTAB buffer works well for fresh (Fig. 5, CT) and ethanol preserved samples, although resulted in an smaller amount of DNA. The combination of CTAB with proteinase K reduces so much the amount of DNA obtained that it is not visible in agarose gels (Fig. 5, CTK). Finally, in Fig. 6, a comparison of preservation and extraction methods is shown. Sponge samples preserved and extracted in/with LBWGH buffer resulted in perfect DNA for PCR amplification. A proposal taken from this work is the use of the LBWGH buffer for the short-time fixation of sponge samples at room temperature, followed by the LBWGH plus proteinase K DNA extraction. Being a simple solution, like RNALater®, the LBWGH should not be a problem to exchange loans by conventional mail. The use of screw capped 2 mL vials is recommended, always using three times more LBWGH buffer than sponge fragments. For long-time fixation in LBWGH buffer, samples should be kept at -20o.C.
Appendix Method 1, Lyses Buffer with Proteinase K (LBWPK): Homogenization in 1:3 (weight:volume) solution of SET buffer (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 was incubated at 55ºC for 1 hr and centrifuged at 3000 g/10 min. The supernatant was extracted once with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and twice with one volume of chloroform. Precipitate DNA from the homogenate by the addition of 2 volumes of ethanol plus 1/10 volume of 3 M sodium acetate pH 5.2, followed by centrifugation at 10000 g/10 min at 4ºC. The pellet was washed in 70% ethanol, air dried, dissolved in sterile water plus 20 μg/ml RNAse A (GIBCO BRL), and incubated for 1 h at 37ºC.
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Fig. 5: DNA quantification and scores from fresh collected samples showing the extraction products from two different individuals of H. heliophila with four different methods 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 Ammonium Bromide (CTAB): Homogenization of sponge fragments in 1:3 (weight:volume) solution of 2% CTAB in 100 mM Tris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl plus 1 µL βmercaptoetanol and 10 µg/µL proteinase K. The suspension was incubated at 50ºC for at least 1 hr and centrifuged at 3000 g/10 min. The supernatant was extracted twice with one volume of chloroform: isoamyl alcohol (24:1). Precipitate DNA from the homogenate by the addition of 0.8 volumes of isopropanol plus 1/10 volume of 3 M sodium acetate pH 5.2, followed by centrifugation at 10000 g/10 min at 4ºC. The pellet was washed in 70% ethanol, air dried, dissolved in sterile water plus 20 μg/ml RNAse A (GIBCO BRL), and incubated for 1 h at 37ºC. Method 3, Lyses Buffer with Guanidine Hydrochloride (LBWGH): Specimens were ground with a rod in a mortar with 1:5 (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 suspension was incubated at 50ºC for 1 hr and centrifuged at 3000 g/10 min. The supernatant was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and nucleic acids were precipitated with 2 volumes of ethanol. The pellet was washed in 70% ethanol and air dried. The dried pellet was dissolved in sterile water plus 20 μg/ml RNAse A (GIBCO BRL) and incubated at 37ºC for 2 h. Method 4, DNAzol®: Homogenization of 25-50 mg fragments in 1 ml of DNAzol® in a hand held homogenizer by applying as few strokes as possible. The homogenate was centrifuged at 3000 g/10 min. The supernatant was transferred to a fresh tube and the DNA precipitated by the addition of 0.5 ml of 100% ethanol per 1 ml of DNAzol® used for the isolation. The DNA precipitate was removed by spooling with a pipette tip or by centrifugation at 10000 g/10 min at 4ºC. DNA pellet was washed twice with 75% ethanol and dissolved in water.
Fig. 6: DNA quantification of total genomic DNA extracted by the LBWGH method. Molecular weight marker are from top to bottom: 23100, 9400, 6600, 4400, 2300, 2000 bp. E96 = sponge specimens preserved at museum collections for long term in ethanol 96%, and L = sponge specimens preserved directly in LBWGH.
Acknowledgements D. M. Braga de Mello is thanked for technical assistance. This work was carried out with financial assistance from Programa Prociência, Sub-reitoria de Pós-graduação e Pesquisa (SR2-UERJ), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de
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Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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