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Direct Amplification and Naoh Extraction Journalof Bryology (2002) 24: 127±131 Directampli® cation and NaOH extraction: two rapid and simple methodsfor preparing bryophyte DNA for polymerase chain reaction(PCR) OLAF WERNER, ROSA MARI Â AROS andJUAN GUERRA University ofMurcia, Spain SUMMARY PCR(polymerase chain reaction) has become one of the most important techniques used in molecular systematics.Generally, the methods applied to isolate DNA for PCR ampli® cation depend on multiple stepsto isolate and clean the ® nalproduct. This involves considerable e ort and time when many sampleshave to be processed and poisonous organic solvents are often needed (phenol, chloroform etc.).In this contribution, two very rapid and simple techniques intended for use in higher plants are shownto be useful in bryophyte molecular biology: direct ampli® cation from plant tissues and the NaOHextraction method. In 15 of the 17 investigated bryophytes (two hepatics and 15 mosses) the trnLUAA intronof the chloroplast DNA was successfully ampli® ed by the direct approach, while the NaOHextraction method gave ampli® able DNA in all 17 species. DNA ampli® ed by both methods wassuccessfully used in cycle sequencing. KEYWORDS:direct PCR, NaOH DNA isolation, trnLUAA intron,bryophyte DNA extraction. INTRODUCTION whenlarge numbers of samples have to be processed, for examplein population genetic investigations or when PCR(polymerase chain reaction)-based techniques are extremelysmall bryophytes are studied. Therefore, we becomingincreasingly popular for studying relatedness of comparedboth methods with several small moss and bryophytetaxa. The sequencing of PCR-ampli® ed DNA hepaticspecies. fragmentsand RAPD (random ampli® ed polymorphic DNA)are two frequently used techniques. The methods thatare generally used for DNA extraction from bryo- MATERIALSAND METHODS phytesare based on those described by Doyle & Doyle Plantmaterial (1987,1990) using CTAB assolvent (cf. Ashton et al., 1994;Boisselier-Dubayle et al.,1995;Capesius & Bopp, Plants of Metzgeriafurcata (L.) Dum., Lophocoleabiden- 1997;Meiû ner et al.,1998;Patterson et al.,1998;De Luna tata (L.)Dum. and Fissidenstaxifolius Hedw. were et al.,1999;Stenù ien, 1999) although other methods, such collectedfrom S.W. Germanyin January 2000. Didymodon asthe SDS extraction method of Edwards, Johnstone & sicculus M.J.Cano,Ros, Garcõ Âa-Zamora & J.Guerra, Ptery- Thompson(1991) are sometimes used (cf. Cox & Hedder- goneurumlamellatum (Lindb.)Jur., Bryumargenteum son,1999). All these methods require a considerable Hedw.,and one sample of Aloinaaloides (Schultz)Kindb. investmentin both time and the chemicals needed. Two werecollected during February 2000 from S.E. Spain. methodshave been published for the very rapid prepara- Othersamples of Aloinaaloides wereherbarium specimens tionof plant material for PCR, although so far no studies ofSpanish origin sampled in the years 1999, 1997, 1993 havebeen published that have applied these techniques to and 1990. Grimmiatrichophylla Grev., Brachytheciumvelu- bryophytes:the direct ampli® cation of DNA from plant tinum (Hedw.)Schimp., Pterogoniumgracile (Hedw.) Sm, tissues(Berthomieu & Meyer,1991; Rogers & Parkes, Homalotheciumlutescens (Hedw.)Robins., Hypnum 1999)and the alkaline isolation of total DNA (Wang, Qi cupressiforme Hedw., Rhynchostegiummegapolitanum &Cutler,1993). Such techniques could be very useful (H.F.Weber& D.Mohr)Bruch, Schimp. Eurhynchium # BritishBryological Society 2002 Received 20 November 2000. Revision accepted 4May 2001 DOI:10.1179/ 037366802125000980 128 O.WERNER,R. M.ROSAND J. GUERRA meridionale (Bruch,Schimp. & W.Gu È mbel)De Not. and membranewas made in two steps using 50 l loftheelution Homalotheciumaureum (Spruce)Robins. were collected buer supplied by the manufacturer. The eluates were duringSeptember 2000 from Sierra Espun Äa,S.E. Spain. thencombined to give a ®nalvolume of 100 l l. NaOH- Additionalsamples of Homalotheciumaureum were extractedor Qiagen-extracted DNA (1 l l)that was then herbariumspecimens collected in the years 1999, 1997, usedin the PCR reactions. At least three independent sets 1994,1991, 1986 and 1981. Tortulamuralis Hedw. and ofreplicate experiments were run for all the extraction Tortularevolvens (Schimp.)G.Roth. were collected methods. duringMarch 2001 from Murcia, S.E. Spain. All the ThePCR reactions were performed in 0.2 ml reaction plantswere stored dry until direct PCR or DNA-extrac- tubesusing 1 UTaqDNA polymerase (Appligene Oncor), tionwas carried out. Herbarium vouchers are deposited 0.2mM dNTPs(Appligene Oncor), 10 mM Tris±HCl, pH at MUB. 8.3(RT), 50 mM KCl and1.5 mM MgCl 2 in 25 l l ®nal volume.The primers were designed to amplify the trnLUAA intronof cp DNA. 10 pmol of primers C andD Preparationof plantmaterial for direct PCR (Taberlet et al.,1991)were used with the modi® cations introducedby Meiû ner et al.(1998)to improve annealing Inthe case of terricolous mosses, the lower part of the inbryophytes. An Eppendorf Mastercycler gradient was plantswas cut o to remove the soil particles adhering to programmed(2 min 94 ¯C,35cycles with 15 sec 94 ¯C, 30 therhizoids. All plants were rinsed with double-distilled sec 50¯C, 1 min 72¯Canda ®nalextension step of 2 min water(DDW) to remove dirt and biological contaminants at 72¯)toperform the ampli® cation reactions. The ampli® - asfar as possible.The bryophytes were then transferred to cationproducts were separated on 9% PAA gels and 1.5ml Eppendorf tubes with sterile DDW and treated in DNAbands were visualized using the silver staining anultrasonic cleaner to remove any remaining foreign methodof Dean & Milligan(1998). Gels were scanned material.Such ultrasonic cleaning of small bryophyte usinga TWAINcompatible scanner (Agfa SnapScan 600) samplesin plastic tubes works well andis routinely witha transilluminationmodule and stored in digital appliedin our laboratory for the preparation of material format. forSEM studies.Clean leaves or thallus fragments of PCRproducts were puri® ed using the QIAquick Puri® - ca 0.2 mm2 wereremoved with sterile forceps and trans- cationKit (Qiagen). Sequencing reactions were carried out ferredto thin-walled 0.2 ml Eppendorf tubes, to which usingthe Biocycle Sequencing Kit of GATC (Konstanz, PCRcomponents were added. In order to investigate the Germany)in an Eppendorf Mastercycler (2 min 95 ¯C, 35 dependanceof the PCR process on the amount of plant cycles30 s95 ¯C, 30 s 55¯C, 1 min 72¯C)andprimers C/ D material,varying amounts of Bryumargenteum plant ofTaberlet. Sequencing reactions were separated in a materialwere used. GATC-1500-systemand transferred to Nylon membranes. Basically,NaOH extraction followed the protocol of Thebiotin-modi® ed ddNTPs of the sequencing kit allowed Wang et al. (1993)with minor modi® cations. One whole thevisualization of the DNA-bands by the application of plantwas put in an Eppendorf tube and 5 l l of 0.5 M astandardprotocol with StreptavidinAlkaline Phospha- NaOHwere added. The plant was then ground with a tase(GATC) and BCIP/ NBT (Aldrich). stainlesssteel needle, which had been cleaned with 0.5 M HCl, untilno larger fragments were visible. An additional 15 l l0.5M NaOHwere then added and grinding was RESULTS AND DISCUSSION continuedfor 1 or2min.The samples were centrifuged at 13,000rpm for 2 minand the supernatant was diluted 1:10 Fig.1 showsan example of the direct ampli® cation of the in100 mM Tris±HCl, pH8.3. When necessary, this dilu- trnLUAA intronof chloroplast DNA. Ampli® cation tionwas used for the preparation of further dilutions with productswere obtained for all species studied with the 100mM Tris±HCl, pH8.3. exceptionof Lophocoleabidentata and Rhynchostegium Forcomparison purposes, DNA was extracted with a megapolitanum .Insome species ( Grimmiatrichophylla , commerciallyavailable kit, which had previously given Brachytheciumvelutinum , Homalotheciumlutescens , and goodresults in our laboratory with higher plants (DNeasy Eurhynchiummeridionale )notall replicates resulted in PlantMini Kit,Qiagen GmbH, Hilden, Germany). This detectableampli® cation products. Rogers & Parkes(1999) kituses silica-gel membranes to bind DNA selectively, observedthat an excess of tissue rather than too little is whichcan then be washed with an appropriate bu er and morelikely to cause problems in direct PCR of higher latereluted. In a modi®cation of the manufacturer’s proto- plants,attributing these problems to leaf matrix compo- col,one plant was ground in 5 l lbuer AP1 in an Eppen- nentssuch as proteins, carbohydrates, chlorophyll, etc., dorftube at room temperature. When no large fragments thatmay act as Taq polymerase inhibitors. Therefore an werevisible, 195 l lofAP1 were added and the Qiagen attemptwas made to reduce the amount of plant material protocolwas followed but with all the bu er volumes, of Lophocoleabidentata tothe minimum that could be withthe exception of the washing bu er, reduced by half. easilyhandled ( ca 25 25 l m)but no ampli® cation was £ The® nalelution of the DNA from the silica-gel observedfor any L.bidentata directPCR reaction. DIRECTAMPLIFICATION AND NaOHEXTRACTION OF BRYOPHYTEDNA 129 Figure 1. Directampli® cation ofthe trnLUAA intronusing bryophyte leaves orthallus fragments. Lane M 50bpladder; 1 Fissidens ˆ ˆ taxifolius; 2 Metzgeria furcata ; 3 Lophocolea bidentata ; ˆ ˆ 4 Didymodon sicculus ; 5 Pterygoneurum lamellatum ; 6 Bryum ˆ ˆ ˆ argenteum; 7 Aloina aloides ,collected 2000; 8 Aloina aloides , 1999; ˆ ˆ 9 Aloina aloides , 1997; 10 Aloina aloides , 1993; 11 Aloina aloides , Figure 3. Inhibitorye ect oflarge amountsof plant
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