The analysis of genetic polymorphisms in groups of organisms leads to an understanding of the processes that structure populations, and, increasingly, DNA polymorphisms are the subject of study

Grahame, J

Simpson, RJ

Wilding, CS

LJMU Research Online (Liverpool John Moores University)

 Simpson, RJ, Wilding, CS and Grahame, J Simple protocol for extracting nuclear DNA from single embryos of a marine snailhttp://researchonline.ljmu.ac.uk/2712/ArticleLJMU has developed LJMU Research Online for users to access the research output of the University more effectively. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LJMU Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain.The version presented here may differ from the published version or from the version of the record. Please see the repository URL above for details on accessing the published version and note that access may require a subscription. For more information please contact researchonline@ljmu.ac.ukhttp://researchonline.ljmu.ac.uk/Citation (please note it is advisable to refer to the publisher’s version if you intend to cite from this work) Simpson, RJ, Wilding, CS and Grahame, J (1999) Simple protocol for extracting nuclear DNA from single embryos of a marine snail. BIOTECHNIQUES, 26 (6). pp. 1050-1052. ISSN 0736-6205 LJMU Research OnlineBenchmarks1050 BioTechniques Vol. 26, No. 6 (1999)DNA in solution were used instead of10.75 µL of ddH2O and 8.5 µL of DNAin solution. The re-amplified elutedband was electrophoresed, gel-purifiedand cloned into a pGEM-T vector(Promega, Madison, WI, USA). Wethen identified clones containing re-combinant plasmids, isolated plasmidDNA and used the insert of an individ-ual clone as a hybridization probe innorthern blot analysis. A single hy-bridizing mRNA of approximately 520bases was identified in the lanes corre-sponding to the CM and, to a lesser ex-tent, the 90-day-old WT hamster ven-tricles (Figure 1C).By using the re-amplified band of in-terest as a hybridization probe againstnorthern blots, we were able to isolate,clone and identify a differentially ex-pressed cDNA despite the fact that nu-merous cDNA species were present inthe original band obtained. The abilityto isolate and clone DNA that has hy-bridized to specific mRNA speciesshould increase the speed of the sec-ondary screening following DD andshould eliminate the possibility thatpositive bands are not discarded simplybecause the specific clone is overlookedin the secondary screening analysis.REFERENCES1.Bauer, D., H. Muller, J. Reich, H. Riedel, V.Ahrenkeil, P. Warthoe and M. Strauss.1993. Identification of differentially expressedmRNA species by an improved display tech-nique (DDRT-PCR). Nucleic Acids Res.21:4272-4280.2.Denovan-Wright, E.M., R.A. Newton, J.N.Armstrong, J.M. Babity and H.A. Robert-son. 1998. Acute administration of cocaine,but not amphetamine, increases the level ofsynaptotagmin IV mRNA in the dorsal stria-tum of rat. Molecular Brain Res. 55:350-354.3.Liang, P. and A.B. Pardee. 1992. Differentialdisplay of eukaryotic messenger RNA bymeans of polymerase chain reaction. Science257:967-971.4.Liang, P. and A.B. Pardee. 1997. Differentialdisplay. A general protocol. Methods Mol.Biol. 85:3-11.5.McClelland, M., F. Mathieu-Daude and J.Welsh. 1995. RNA fingerprinting and differ-ential display using arbitrarily primer PCR.Trends Genet. 11:242-246.6.Sambrook, J., E. Fritsch and T. Maniatis.1989. Molecular Cloning: A Laboratory Man-ual, 2nd ed. CSH Laboratory Press, ColdSpring Harbor, NY. 7.Utans, U., P. Liang, L.R. Wyner, M.J.Karnovsky and M.E. Russell. 1994. Chroniccardiac rejection: identification of five upregu-lated genes in transplanted hearts by differen-tial mRNA display. Proc. Natl. Acad. Sci.USA 91:6463-6467.Address correspondence to Dr. HaroldA. Robertson, Laboratory of MolecularNeurobiology, Department of Pharmacolo-gy, Dalhousie University, Halifax, NovaScotia, Canada. Internet: har1@is.dal.caReceived 9 November 1998; accepted8 March 1999.Eileen M. Denovan-Wright,Susan E. Howlett and HaroldA. RobertsonDalhousie UniversityHalifax, Nova Scotia, CanadaSimple Protocol forExtracting Nuclear DNAfrom Single Embryos of aMarine SnailBioTechniques 26:1050-1052 (June 1999)The analysis of genetic polymor-phisms in groups of organisms leads toan understanding of the processes thatstructure populations, and, increasingly,DNA polymorphisms are the subject ofstudy. Workers may be interested in or-ganisms as research tools for under-standing either evolutionary processes,the impact of pollutants or the sustain-ability of harvesting regimes.  Marineanimals figure prominently in all ofthese fields, but the extraction of DNAthat is suitable for polymerase chain re-action (PCR) from small marine em-bryos is complicated by the small quan-tities of tissue initially present. A recentinvestigation using our model organism[Littorina saxatilis (Olivi)] recommend-ed the use of the relatively expensiveChelex (12). The technique we describehere extracts DNA from individualwhole embryos by incubating them in asimple lysis buffer, thus allowing DNAfrom individual embryos to be used inboth mitochondrial and nuclear investi-gations (nuclear investigations need ahigher-quality DNA extraction productthan do mitochondrial investigations).This single-step procedure is relativelyinexpensive and yields DNA that is di-rectly suitable for PCR, whereas extrac-tions using toxic reagents (8,14) and in-volving several handling steps (7,16)are cumbersome when very smallamounts of tissue are involved.The embryos of the intertidal snailLittorina saxatilis are found in a moth-er’s brood pouch (13). A method ofextracting DNA from the adults is a phe-nol/chloroform-extraction (6); however,this is a difficult technique to applywhen very small amounts of tissue areinvolved. An alternative is simply to am-plify after adding an embryo to the PCRmixture, but this often fails to producereliable amplifications (see Figure 1). Inthis paper, using both nuclear and mito-chondrial PCR primers, we compare thesuccess rates for two different extractiontechniques: (i) directly adding wholeVol. 26, No. 6 (1999) BioTechniques 1051embryos to the PCR mixture, and (ii)adding products that are recovered fromembryos using the lysis buffer.We have used a buffer that wasslightly modified from that describedby Higuchi (10), which has been usedfor extraction of single Caenorhabditiselegans (1) and blood biopsies of adultMytilus (a technique unsuitable for verysmall marine animals; Reference 5).For extraction, a single embryo (be-tween 0.4 and 0.7 mm in diameter; Ref-erence 11) was added to 10 µL of ex-traction buffer (50 mM KCl, 10 mMTris-HCl, pH 8.3, 2.5 mM MgCl2,0.01% gelatin, 0.9% Tween 20 and 10mg/mL proteinase K) in a 0.5-mL mi-crocentrifuge tube. Mineral oil (20 µL)was added to each tube before incuba-tion. We used a one-step incubation/de-naturation protocol, incubating thesample for 60 min at 65°C followed bydenaturation for 15 min at 94°C. Theextracted samples can be kept frozen at-20°C until needed for use in PCR—material stored for five months hasbeen successfully amplified. The buffercan be made in advance and stored at-20°C indefinitely, although fresh pro-teinase K must be added before use.L. saxatilis embryos develop in thematernal brood pouch from thesingle-cell zygote to a shell-bearing ju-venile that hatches. The extraction tech-nique has allowed successful amplifica-tions using embryos from the earlyzygote stage to the early shell stage(proteinaceous shell only) and usingboth frozen (-70°C) and fresh embryos.Using this procedure, DNA can be ex-tracted from large numbers of singleembryos quickly and efficiently, and be-cause only one tube is used from intro-duction of material until removal forPCR, the chances of contamination orloss of material are low. The lysis proce-dure yields sufficient extract for multi-ple separate PCRs (2.5 µL extract each).This technique is an advantage overthose described by  Coffroth and Mu-lawka (3) and Geller et al. (9),  wheremore complicated protocols involvedbuffers containing hexadecyltrimethyl-ammonium bromide (CTAB), and byCôrte-Real et al. (5), which yielded suf-ficient extract from a single mussel lar-va for only one PCR.Figure 1 shows results from use ofthis extraction protocol. In this trial, weamplified DNA from targets preparedusing either the lysis protocol or simplyfrom native embryos placed into thePCR mixture. We amplified productsfrom the calmodulin region of the Litto-rina nuclear genome. The primers usedwere the universal CAD2 and CAD3(4). Using the lysis protocol, 76% (of41 amplifications) were successful vs.28% (of 43 amplifications) when no ly-sis buffer was used. Only 39% (of the31 successful amplifications) from thelysed samples yielded faint bands, indi-cating weak amplification; whereas,92% (of the 12 successful amplifica-tions) in the samples that were addeddirectly to PCR mixtures yieldedbands. In a trial where the amplificationtarget was mitochondrial cytochrome b,using primers CytbFOR (5′-TTCC-CGCACCTTCAAATCTT-3′) and Cyt-bREV (5′-AGGGAACTTTTTCTCCATCTCTGT-3′) (C. Wilding unpub-lished) both lysed and non-lysed sam-ples produced similar results with ap-proximately 80% (of 40 amplifications)amplification, and all bands showingstrong amplification.Nuclear DNA is associated withcomplex protein structures (nucleo-somes) (2), which have to be brokendown during replication. If DNA doesnot dissociate from this molecule, it isunavailable to the primers in a PCR, andthus amplification does not occur. Therelative ease of release of mitochondrialDNA into the solution where it is thenavailable for PCR rests on three factors:(i) it occurs in an organelle other thanthe nucleus, (ii) it does not associatewith nucleosomes and (iii) it has a highcopy number. Extracts therefore containhigher concentrations of mitochondrialthan of nuclear DNA (15). The ob-Figure 1. Genomic DNA isolated from L. sax-atilis embryos. Lanes 1 and 18, Lambda EcoRI-HindIII marker DNA (Advanced BiotechnologiesLtd., Epsom, Surrey, UK); lanes 10–17, calmod-ulin PCR products from samples added to thePCR mixture. Lanes 2–9, calmodulin PCR prod-ucts from samples that underwent the lysis proto-col. Resolution on a 1% agarose gel visualizedwith ethidium bromide.Benchmarks1052 BioTechniques Vol. 26, No. 6 (1999)served difference in efficiency of ampli-fication between nuclear and mitochon-drial targets, depending on whether ly-sis was used or not, is probably due tothe relative ease with which quantitiesof DNA from rupturing mitochondriaare released, as compared with the re-lease of more sequestered DNA fromthe cell nucleus.REFERENCES1.Barstead, R.J. and R.H. Waterston. 1991.Vinculin is essential for muscle function in thenematode. J. Cell. Biol. 114:715-724.2.Brown, T.A. 1998. Genetics a Molecular Ap-proach. Chapman and Hall, London.3.Coffroth, M.A. and J.M. Mulawka III. 1995.Identification of marine invertebrate larvae bymeans of PCR-RAPD species-specific mark-ers. Limnol. Oceanogr. 40:181-189.4.Côrte-Real, H.B.S.M., D.R. Dixon andP.W.H. Holland. 1994. Intron-targeted PCR: anew approach to survey neutral DNA polymor-phism in bivalve populations. Mar. Biol.120:407-413.5.Côrte-Real, H.B.S.M., P.W.H. Holland andD.R. Dixon. 1994. Inheritance of a nuclearDNA polymorphism assayed in single bivalvelarvae. Mar. Biol. 120:415-420.6.Crossland, S., D. Coates, J. Grahame andP.J. Mill. 1993. Use of random amplified poly-morphic DNAs (RAPDs) in separating two sib-ling species of Littorina. Mar. Ecol. Prog. Ser.96:301-305.7.Evans, B.S., R.W.G. White and R.D. Ward.1998. Genetic identification of asteroid larvaefrom Tasmania, Australia, by PCR-RFLP. Mol.Ecol. 7:1077-1082.8.Gasser, R.B., N.B. Chilton, H. Hoste and I.Beveridge. 1993. Rapid sequencing of rDNAfrom single worms and eggs of parasitichelminths. Nucleic Acids Res. 21:2525-2526.9.Geller, J.B., J.T. Carlton and D.A. Powers.1994. PCR-based detection of mtDNA haplo-types of native and invading mussels on thenortheastern Pacific coast: latitudinal pattern ofinvasion. Mar. Biol. 119:243-249.10.Higuchi, R. 1989. Simple and rapid prepara-tion of samples for PCR, p. 31-38. In H.A. Er-lich (Ed.), PCR Technology. Stockton Press,NY.11.Hull, S.L., J. Grahame and P.J. Mill. 1996.Morphological divergence and evidence for re-productive isolation in Littorina saxatilis(Olivi) in northeast England. J. Mol. Stud.62:89-99.12.Mikhailova, N. and K. Johannesson. 1998. Acomparison of different protocols for RAPDanalysis of Littorina. Hydrobiologia 378:33-42.13.Reid, D.G. 1996. Systematics and Evolution ofLittorina. The Ray Society, London.14.White, L.R., B.A. McPheron and J.R. Stauf-fer, Jr. 1994. Identification of freshwater mus-sel glochidia on host fishes using restrictionfragment length polymorphisms. Mol. Ecol.3:183-185.15.Wilson, A.C., R.L. Cann, S.M. Carr, M.George, U.B. Gyllensten, K.M. Helmby-chowski, R.G. Higuchi, S.R. Palumbi, E.M.Prager, R.D. Sage and M. Stoneking. 1985.Mitochondrial DNA and two perspectives onevolutionary genetics. Biol. J. Linn. Soc.26:375-400.16.Wray, C.G., J.J. Lee and R. DeSalle. 1993.Extraction and enzymatic characterization offoraminiferal DNA. Micropaleontology39:69-73.This study was funded in part by Con-tract No. MAS3-CT95-0042 from the Euro-pean Union. We thank the School of Biologyat the University of Leeds for the use oftheir facilities and Dr. I.A. Hope and his re-search group for their assistance. Addresscorrespondence to Mr. Robert J. Simpson,School of Biology, The University of Leeds,Leeds, UK. Internet: bgyrjs@leeds.ac.ukReceived 19 November 1998; accepted1 March 1999.Robert J. Simpson,Craig S. Wilding andJohn GrahameThe University of LeedsLeeds, UKExtracting High-QualityDNA from Shed ReptileSkins: A SimplifiedMethodBioTechniques 26:1052-1054 (June 1999)Molecular studies involving reptilesoften overlook shed skins as a sourcefor high-quality DNA. In most cases,tissues or blood samples are preferredby researchers, but the process of sam-pling for these tissue types can beharmful or otherwise adversely affectthe animals involved. While reptilebreeders or zoological institutions are apotential source of specimens, mostwill likely decline requests for samplesif the sampling will harm their prizedanimals. Generally, breeders and cura-tors would be much more amenable topart with a shed skin—which is some-thing they usually discard anyway.Figure 1. Agarose gel depicting the quality ofthe genomic DNA extractions produced by thismethod (lanes 1–5 and 13). Subsequent cy-tochrome-b PCRs generated from these extrac-tions (lanes 7–11 and 14) using the LGL765primer (5′-GAAAAACCAYCGTTGTWATTC-AACT-3′) of Bickham et al. (2) and the H15919primer (5′-GACCCAKCTTTGRYTTACAAG-GACAA-3′) from this study are also shown.These primers produce a product that is approxi-mately 1160 bp in length. Extractions from thedifferent species are as follows: L. mexicana, lane1; L. g. floridana, lane 2; L. p. pyromelana, lane3; L. alterna, lane 4; L. z. zonata, lane 5. Lane 6contains a 1-kb DNA Ladder (Life Technolo-gies). Lanes 7–11 contain the same species (inthe same order) as lanes 1–5. Lane 12 contains a100-bp DNA ladder (Life Technologies). Lanes13 and 14 contain extracted DNA and resultingPCR product, respectively, from the day gecko P.madagascariensis grandis.

Simple protocol for extracting nuclear DNA from single embryos of a marine snail

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Simple protocol for extracting nuclear DNA from single embryos of a marine snail

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