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rSOL\TIO~T OF PlANT DNA FROM FRESH TrSSUE, .

'T he isolation of high rnolccular weightI DNA that is suitablc for digestion

with restriction endonucleases can' bea serious stumbling block to progrcss

i rnolccular srudicss of many types. Plants areP Irticularly notorious for their intractability withrr,any isolation procedures. Furthcrmorc, a pro-ccdurc that works with one plant group willof .cn fail rniscrably with others, an outcome that; not uncxpccted givcn the diversity of plants~ 1 :l their secondary compounds.

Oldcr methods for DNA isolation frompl lo (S require large arnounts of rissuc due ro lowyicl-, , clcarly a drawback when DNA must beiso l: -:d from n u rn cr ou s small individuals .

• Furrr; '. more: merhods rhat rely on CsCl gradi-c n t s e time consuming and expensive.Forrun. :Iy, a diversity of lcss expensive proto-cols hav : bccn dcvclopcd that are charactcrizcdby high .' elds of D lA frorn small amounts oftissuc. S,, -c ral of t h csc (1·4) use hcxade-cyl-rimcrhy: .mmoniurn brornidc (CTAB). Hercwc describe xur versión of a proccdure for isola-tion of DN! from frcsh plant tissuc using CTAB(2), which i: a modification of thc method ofSaghai-Marcof ct al. (4) with lyophilizcd lcavcs.

This CTAB mcthod for fresh tissue has beenuscd succcssfully in our laboratory on a wide tax-onomic sampling of plant farnilics, includingborh monocots (e.g., palrns , grasses, scdgcs,orchids) and dicots (e.g., walnurs, hickories,oaks, bccches, lcgumcs, applcs and rclativcs, sax-ifragcs, lobelias, brassicas, portulacas), as well asconifers and ferns. The rncthod also has bcenuscd successfully with rccently dried (up to 2ycars), prcsscd lcavcs (3) and outside the planrkingdorn with insccts (5).

PROTOCOL

1. I'rchcat 5 ro 7.5 mi ofCTAB isolation buffer,[2% (wyv) CTAB (Sigma), lA M NaCI,]0.2% (v/v) 2-mereaptoethanol, 20 mMEDTA, 100 mM Tris-HCI, (pH 8.0)J in a .3D-mi glass ~entrifuge tube to 60°C in awater barh.

2. Grind 0.5 to 1.0 g of frcsh, lcaf tissue to apowdcr in liquid nirrogcn in a chille~ morrar

FOC'/S 12 :-J 1I M 11 E Il

.and pcstlc.3. Scrape thc powdcr directly into prchcarcd

buffer and swirl gcntly to rnix.Commen·t.: Alternatively, fresh tissuc may beground in. CTAB isolarion buffer at 60°C in aprchcatcd rnortar.4. Incubatc thc sarnplc ar 6GoC for 30 rnin with

optional occasional gentle swirling.5. Extract once with chloroform-isoamyl alco-

hol (24:1; v:v), mixing gcntly but thor-oughly.

6. Centrifuge (1600 xg) in a clinical cemrifuge(swinging bucket rotor) ar room tcrnpcra-ture td the concentra te phascs.

Commcnt:" Gencrally thc aqueous phasc \ViII beclear following ccntrifugation, but this is noralways the case.7. Rcrnove thc aqueous phase with a wide-borc

pipet, transfer to a clean, giass ccntrifugctube, add 2/3 volurnes of cold isopropanol,and mix gently to pre.cipitate the nuc1cicacids.

Comment: In so me cases, this stage yiclds largcstrands of nucleic acids that can be spooled witha glass hook. Gcnerally, the samplc is cithcr Iloc-culcnt or clo dy. If no cvidcncc of prccipitationis observed at this stagc, thc samplc rnay be lcfrat room tc m p c r a tur e for scvcr a l hours to Iovcrnight. This is onc convcnicnt stopping place,in fact, whcn many samples are being prepared.In ncarly all cases, thcrc is cvidcncc of precipita-tion 'aficr the samplc has bccn allowed to scttlcout in this manner.8. Rccover the nucleic acid by onc of rhe fol-

lowing oprious:a. If strands of DNA are visible, spool rhc

nucleic acids with a glass hook and trans-fer to lOto 20 mi of wash buffer [76%(v/v) c t h a no l , 10 mM a m m o n iu macctatc l. .

b. If the DNA appcars flocculcnt, cenrrifugcat (500 x g) for 1 ro 2 mino Gcntly pouroff as much of thc supcrnatc as possiblcwithout losing the precipitatel which willbe adiffuse and very loose pella. Addwash buffer directly to the pcllct and swirlgently to resuspcnd the nuclcic acids.

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fIGURE 1. DNA ísolated from plants by the erAn procc:durc:. Sarnplcs wc:rc subjccrcdlO clccrrophorcsis on a 0.7% (w/v) agarosc: gel in Tris-borarc EDTA buffer. unes: 1, A/1l11f

(aldcr); 2, Bolussfra (a lcgumc); 3, Splunostylis (a lcgumc); 4, Clnytonia (spring bcaury); S,Salacca (a palrn); and 6, undigcstcd bacteriophagc A DNA. Molccular wcight standard is AD~JA digcsrcd with Hind Ill. DNA shown in lancs 3 and 5 was isolatcd using rhe liquidnirrogcn procedurc; rhe rcmaining sarnples were isolatcd without liquid nirrogcn.

I 4

I

c. If the preclpltate is nor obvious, ccn-trifuge the solution at higher spccds. ThiswiIl generalIy result in a hard pcllet or,with smalI arnounts, a film on thc bottomof thc rubc that may contain more irnpu-ritics. Such pcllcts are difficult to wash,and may rcquire stirring with a glass rodto promete washing.

Comment: Thc nucleic acids generalIy becomemuch whiter when washed, thoug~ sorne colormay srill remain.9. Cenrrifuge (1600 x g for 10 min) or spóol

the nucleic acids after a minimum of 20 minofwashing.

Comment: The wash stcp is another convenientstopping point. Samplcs can be lcft at roomtempcraturc in wash buffer for at least two dayswirhout noticcable problcrns.10. Pour off the supernatc carefuIly and aIlow

the pcllet to air dry briefly ar room tcrnpcra-turc,

11. Resuspend rhc pcllct in 1 mi TE [10 mMTris-HCI (pH 7.4), 1 mM EDTA].

Comment: Gel clcctrophorcsis of thc nucIeicacids at this stcp often rcvcals the presence ofvisible bands of rRNAs, as weIl as high rnolecu-lar weight DNA. Alrhough we commonly con-tinue with steps 12 through 15, the DNA at thisstage is generalIy suitable for rcstriction diges-tion. If the DNA is uscd at this stagc, the pellcrsshould be dried more thoroughly than indicatcd

in step 10.12. Add RNase A to a final conccnrration of 10

¡.tgjml and incubaré 30 rnin ar 3rc.13. Dilutc thc sample wirh 2 volurncs of disrillcd

water or TE and ad d 7.5 M a m rno n iuruacctatc (pH 7.7) to a tinJI conccntrarion of2.5 M and 2.5 volurncs of cold crhanol.Gently rnix to prccipirarc the DNA.

Comment: DNA ar this srage usually appearsc lca n e r t h a n in the pr c vio u s p rccip itar io n.Dilution with water or TE is hclpful, as wc havcfound thar prccipirarion from a 1 011 total vol-urnc often produces a gclarinous prccipirarc tharis difficult to pellcr and dry adcquarcly.14. Centri thc DNA 10,000 x g for 10 min ~toI

. fi d .tu- o~~~10 a re n crare ccntn ge. .-", \.rt' ,.J1"/'15. Air dry rhc sa rnp lc a n d rcs uspcn d In a n \">t'roappropriatc amount ofTE.

RESULTS AND DISCUSSIONThis procedure yiclds total DNA, both

nuclear and chloroplasr sequcnces (dara notshown). Thc DNA is gcncrally higb molccularwcight (figure 1), although ofren sornc lowmolecular weighr fragments are obscrvcd. Thediffere nce in DNA qualiry bcrwccn samplesshown is rcprcscntativc of most plant groups.The liquid nitrogen proccdurc tc nSistenflY)yiclds DNA of higher average molccular weightthan docs thc rncthod in which fresh rissuc isground directly in buffer. Howcvcr, for mosrapplications, such as screening largc nl!rr~crs ofindividuals, wc routincly use tte~h-grouod sarn-pies.

Yiclds using thcsc rncthods often ap~roach1 mg/g fresh tissuc, although this is srI'onglydependent on both thc age and qualiry of thetissue and on the spccics uscd:....Quaneification bY)absorbance at 260 nrn gcncrally gives unrcliablcresults, presumably due to inrcrferencc of resid-ual CTAB in rhc samplcs.

Although thc basic procedure dcscribcdherc has bccn uscd with many diffcrcnt plantgroups, modifications may improvc the qualiryor yield of DNA in sornc planr group~. Forexarnplc, in planes with high concentrarions of Iphcnolic compounds, such as oaks and walnuts,1% (w jv) polyvinylpyrollidine (PVP-40) hasbcen added to the isolation buffer wirh succcss-fuI rcsults (unpublished observarions). Forplanrs c,ontaining high polysaccharide lcvclsandjor glutinous sap, which ofren yicld very vis-cous grindates (eg., Onagraceae, brorncliads ),

¡:OC.US 12 NUM/lER

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•C$$ful isol;ations have been achicved by sim- \ply increasing thc CTAB perccntage to 3% I(w/v) or higher (6).

In sornc cases, DNA obtaincd by this proce-dure is furthcr purified by one or two ultraccn-trifugation stcps in CsC!. This modificationcombines the advantagcs of the high yiclds rou-tincly achicvcd by the CTAB isolation method ACKNOWLEDGEMENTSwith the presumably grcatcr purity of CsCI This work was supportcd in part by grantsmcthods. In many protocols, however, we use) from thc National Scicnce Foundation (BSR-chis CTAB proccdurc as a substitute for CsCI 8516630 and BSR-8805630). We thank Karcngradicnts. For cxamplc, chloroplast DNA isola- Hansen and Dan Potrcr for providing DNArions may be pcrforrncd more rapidly and with samplcs.no apparcnt dctrimcntal cffect by following pub-lishcd protocols for chloroplast DNA isolation(7, 8) up to thc point of obtaining a fractioncnrichcd for chloroplasts or DNA. An cqual vol-urnc of preheared CTAB isolation buffer is then 2.added, and thc protocol given here is followed.

The method describcd herc is readily madi- 3.ficd for very small amounts of fresh tissue. We 4.~:.\ve perforrncd P?pulation surveys with DNA)trom ovcr 200 individual plants, often using0.01 to 0.1 of fresh tissue per plant (9). In such 5.cases, thc Iiquid nitrogcn is omitted. Grinding, 6.incubation in isolation buffer, chloroforrn- 7.isoamyl alcohol cxtraction, and ccntrifugationmay all be perforrned in a single microcentrifuge 8.tube. The DNA, in this case, is used after thcfirst prccipitation. DNA also may be isolatcd 9.from dried sceds; howevcr, the large amounts ofstarch and protein in sccds often rcquirc addi-tional cxtraction with organic solvcnts and fur-ther purificaticn.

CONCLUSIONIn conclusion, this rncthod has proven ea be

a useful addition to thc many DNA isolationprotocols that are now available for planes. lesvcrsatiliry, spccd, and low cost have rnadc ir thcprocedure of choice in our lab and clscwhcrc.

REFERENCES1. Rogers, S.O. and Bcndich, A.J. (1985) Plant Mo/.

Bici. 5, 69.Doylc, ].J. and Doylc J.L. (1987) Pbytochcm, Bu/l.19, 11.

Doyle, J.'} and Dickson, E.E. (1987) Taxon 36, 715.

Saghai-Maroof, M.A., Solirnan, KM., Iorgcnscn,RA and Allard, R..w. (1984) Proc. Natl. AcJUi. SÚ

USA 81, 8014.

Boyce,T., personal comrnunication.Sytsrna,K and Srnith, J.P.. personal communicaáon.Calie, P.J. and Hughes, KW. (1987) Plant Mof. Bio/.Rcporter 4, 206.Bóokjans, G., Srurnmann, B.M. and Hcnningscn,KW. (1984) Anal. BiodJCm.141, 244.Doyle, J.]. and Doylc, J.L. (1988) Amer. ]. Bot. 75,1238.

FO<:US 12 NUMRERI S