Zhanguo Xin, Jeff P. Velten,Melvin J. Oliver, and John J. BurkeUSDA-ARS, Lubbock, TX, USA

ABSTRACT

PCR has become one of the most popu-lar techniques in functional genomics. Pro-jects in both forward and reverse geneticsroutinely require PCR amplification ofthousands of samples. Processing samplesto extract DNA of sufficient purity for PCRis often a limiting step. We have developed asimple 96-well plate-based high-throughputDNA extraction method that is applicable tomany plant species. The method involves asimple incubation of plant tissue samples ina DNA extraction buffer followed by a neu-tralization step. With the addition of a mod-ified PCR buffer, the extracted DNA en-abled the robust amplification of genomicfragments from samples of Arabidopsis, to-bacco, sorghum, cotton, moss, and evenpine needles. Several thousand DNA sam-ples can be economically processed in asingle day by one person without the use ofrobotics. This procedure will facilitatemany technologies including high-through-put genotyping, map-based cloning, andidentification of T-DNA or transposon-tagged mutants for known gene sequences.

INTRODUCTION

The near completion of sequencingof the Arabidopsis and rice genomeshas greatly facilitated research in func-tional genomics (1–3). For example,map-based cloning in Arabidopsis hasbecome routine because of the identifi-cation and generation of tens of thou-sands of randomly distributed polymor-phic DNA markers, made possible bythe sequencing of the Arabidopsisgenome (http://www.arabidopsis.org/cereon/) (4). It has now become feasibleto conduct the large-scale identificationof insertion-tagged mutants for knowngene sequences within genomes and togenerate known mutations in designat-ed genes using the technique targetedinduced local lesions in genomes(known as TILLING) (5,6). However,such technologies are hampered by theneed for processing thousands of reac-tions that are generally required toachieve success. Thus, a primary limit-ing step in all of these procedures is thepreparation of PCR-quality genomicDNA from thousands of samples.

We have devised a simple, low-cost,high-throughput method to prepare ge-nomic DNA for PCR amplification.With this method, a single person canprocess several thousand plant samplesfrom genomic DNA preparation toPCR analysis in a single day. Moreover,this method has the versatility to enableits use with a wide range of plantspecies including previously PCR-re-calcitrant tissues such as pine needles.

MATERIALS AND METHODS

Reagents

Polyvinylpyrrolidone (PVP-40) withan average molecular weight of 40 kDaand BSA fraction V were purchasedfrom Sigma (St. Louis, MO, USA). BSAfrom New England Biolabs (Beverly,MA, USA) that comes with restrictionenzymes could also be used. Hot-StartTaq DNA polymerase was purchasedfrom Qiagen (Valencia, CA, USA).

Solutions and Buffers

Buffer A (100 mM NaOH, 2%Tween® 20) must be made fresh from10 M NaOH and 20% Tween 20 stocksolutions just before use. For buffer B[100 mM Tris-HCl (Sigma), 2 mMEDTA], the pH is about 2.0; it is unnec-essary to adjust the pH of buffer B.

Plant Materials

Arabidopsis and tobacco weregrown in a growth chamber at 21°Cwith 100 µmol quanta/m2 constant lightin a commercial potting mixture (Sun-shine No.1). Sorghum (Sorghum bicol-or) and cotton (Gossypium hirsutum)were grown in a field on site. Moss(Tortula ruralis) tissues were takenfrom cultures grown from spores onsterilized MS medium. Pine needleswere collected from an ornamentalAfghanistan pine tree (Pinus eldarica)growing on site.

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High-Throughput DNA Extraction Method Suitable for PCRBioTechniques 34:820-826 (April 2003)

Primers

All the primers were designed withPrimer3, free software available on-line (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/). The primersfor Arabidopsis marker III_6.5 are 5′-AGAAGGAAACACATTTACGGCT-AT-3′ and 5′-TGATTCTGGTAACA-GGAAATTCAT-3′. The primers forArabidopsis marker T4C21 are 5′-CGGCTTGATCTCCATTGATT-3′ and 5′-TGGAGAGACCCATTTTGCAT-3′.The primers for GFP are 5′-ATCCC-ACTATCCTTCGCAAGAC-3′ and 5′-GCGCTCTTGAAGAAGTCGTG-3′.The primers for a sorghum drought-inducible expressed sequence tag are5′-GGCCATTTTTGGTAAGCAGA-3′and 5′-GTTGATTCGGCAGGTGAG-TT-3′. The primers for cotton Cel A1are 5′-GGATCTGCACCCATCAATCT-3′ and 5′-GCAAAGAGATGGGCTG-AAAC-3′. The primers for a mossrehydrin Tr288 are 5′-GCCCATGCCG-ATAGCGTCCTTAGCC-3′ and 5′-CG-TCGGCATGGGCCCCAAC-3′. Theprimers for pine trans-cinnamate-4-hy-droxylase are 5′-TGTGGTGTCATCG-CCGGATCT-3′ and 5′-CGGAGGAA-GAGCGGGTCGTC-3′.

PCR Conditions

The total PCR volume is 20 µLcontaining 2 µL 10× Qiagen PCRbuffer, 1 µL DNA template, 1.5 mMMgCl2, 0.2 mM each dATP, dCTP,dGTP, and dTTP, 0.25 µM each for-ward and reverse primer, 0.1% BSA(w/v), 1% PVP (w/v), and 0.5 U Hot-Start Taq DNA polymerase. Amplifi-cations were carried out with thermal

cyclers (MJ Research, Waltham, MA,USA). The initial step of 95°C for 15min was followed by 40 cycles of94°C for 15 s, 56°C for 15 s, and 72°Cfor 1 min 30 s, and 1 cycle of 10 minat 72°C. PCR products were analyzedby electrophoresis on 4% agarose gelsin 0.5× TBE.

RESULTS

A Simple High-ThroughputProcedure

Most DNA extraction proceduresrequire extensive maceration of planttissues, which limits their adaptation toa high-throughput platform (7–9). Tocircumvent this limitation, we testedseveral commonly used detergents todetermine their ability to facilitate therelease of genomic DNA from intacttissues of sufficient purity for effectiveand dependable PCR amplification.Two detergents were examined for thisstudy; Tween 20 and Triton® X-100,both commonly used in Taq DNA poly-merase storage buffers and thus lesslikely to result in inhibition of amplifi-cation reactions.

Tween 20 had no apparent effect oneither the efficiency of the PCR ampli-fication or its specificity at concentra-tions up to 2% (v/v) of the PCR mix-ture (result not shown). In contrast,Triton X-100 decreases the specificityof PCR amplification at equivalentconcentrations. From this data, Tween20 was chosen as a possible plant tissuedisruption agent for the passive releaseof genomic DNA for PCR.

Inclusion of 2% Tween 20 in the ex-traction buffer was determined to be ef-ficient at releasing sufficient genomic

DNA from intact plant tissues for usein PCR with no need for maceration.The use of a detergent as a passiveDNA-release agent meant that crosscontamination between samples couldbe avoided with minimum effort. Thisfactor allowed for the use of 96-wellPCR plates to generate high-throughputprocedures for DNA preparations.Table 1 outlines this procedure. Thecomplete procedure, from the collec-tion of plant tissue to the establishmentof PCR in a single 96-well plate, can beachieved in less than 2 h. In addition,for greater output, multiple plates canbe processed simultaneously. As a re-sult, several thousand plant samplescan be processed and the DNA fromthem subjected to PCR in a single daywith no need for either tissue macera-tion or robotic techniques. Figure 1shows a typical Arabidopsis mappingexperiment. The success rate of thisDNA extraction method was over 95%.

BSA and PVP Added in PCRMixture Counteract PCR Inhibitors

A common complication in the useof crude plant extracts for PCR analysisis the presence of Taq DNA polymeraseinhibitory compounds that often resultin the complete lack of amplification oftarget sequences. As a result, DNA ex-traction methods based on simple alka-line lysis work only for a limited num-ber of plant species (7,10). In thisstudy, we tested our detergent-based al-kaline DNA extraction procedure forits susceptibility to release inhibitorycompounds along with the desired ge-nomic DNA from several differentplant tissues. Additionally, proceduresto negate or reduce the observed PCRinhibition were tested.

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1. Transfer sample tissue (approximately 30 mm2) to 96-well plates.

2. Add 50 µL buffer A.

3. Incubate for 10 min at 95°C.

4. Add 50 µL buffer B.

5. Mix at moderate speed.

6. Aliquot PCR mixture to 96-well plates at 20 µL/well.

7.Transfer approximately 1 µL DNA from DNA plates to PCR plates with a 96-pin applicator.

Table 1. Procedure of the High-Throughput DNA Extraction Method

Figure 1. A typical mapping result. DNA pre-pared from F2 plants derived from cross betweenArabidopsis thermal sensitive mutant ts02 (RLD)and a wild-type Arabidopsis Landsberg erecta.The DNA was amplified with marker III-6.5. Thesize for RLD is 281 bp; the size for Ler is 255 bp.The fragments were separated on a 4% agarosegel with 0.5× TBE.

Arabidopsis genomic DNA pre-pared with our method contained noapparent inhibiting compounds as toadversely affect the efficiency of PCR(Figure 2). Arabidopsis genomic DNA(10 ng) purified by step gradient ofCsCl [a gift from Jeff Velten (USDA-ARS, Lubbock, TX, USA)] was usedto compare PCR efficiency with 1 µLcrude genomic DNA preparation in 20-µL reactions. As shown in lanes 4–7 inFigure 2, both DNA samples yieldedstrong amplification. Purified genomicDNA produced slightly stronger sig-nals than crude preparations. However,similar efficiency of amplification ofcrude Arabidopsis genomic DNA wasachieved with or without the presenceof BSA/PVP to counteract the inhibit-ing compounds that may be present inthe crude preparations (Figure 2, lanes6 and 7).

To determine if other plant extractscontain PCR inhibitors and how to re-lease the inhibition, 1 µL crude DNAextracts from each of the following tis-sues, Arabidopsis leaves, tobaccoleaves, sorghum leaves, cotton leaves,moss gametophytes, and pine needles,were added separately to a 20-µL reac-

tion containing the 1 µL crude Ara-bidopsis DNA. Primer pairs for Ara-bidopsis marker T4C21 (see Materialsand Methods section for primer se-quences) were used to amplify the Ara-bidopsis genomic DNA in the presenceof crude DNA preparations from vari-ous plants described above. Followingamplification, the mixture was subject-ed to agarose gel electrophoresis to an-alyze the PCR products. As shown inFigure 2, the crude DNA extract fromcotton leaves (lanes 12 and 13) andpine needles (lanes 16 and 17) con-tained potent PCR inhibitors that se-verely inhibited the amplification ofmarker T4C21.

Many additives or facilitators havebeen used to release Taq DNA poly-merase from the inhibition by compo-nents present in crude DNA prepara-tions from animal and plant tissues(11–13). We tested many of these addi-tives and found that inclusion of a com-bination of 0.1% (w/v) BSA and 1%(w/v) PVP (average molecular weight40 kDa) in the PCR mixture effectivelyreleased the inhibition of Taq DNApolymerase that was generated by theaddition of crude cotton and pine nee-

dle extracts (Figure 2). Subsequently,we tested if the presence of the BSAand PVP could also release inhibitionwhen species-specific primers wereused to amplify from the crude prepara-tions containing the respective genom-ic DNA. The crude genomic DNApreparations could be used in amplifi-cations using species-specific primersfor Arabidopsis, transgenic tobacco,sorghum, and moss, regardless of thepresence of BSA and PVP in the reac-tions (Figure 3). However, the additionof BSA and PVP to the PCR mixture isrequired to amplify efficiently samplescontaining crude DNA preparationsfrom cotton leaves or pine needles.

DNA Prepared by the DescribedMethod Is Compatible withFluorescence-Labeled Primers

Most commercially available high-throughput genotyping technologies re-quire the use of fluorescence-labeledprimers for PCR amplification. The useof such primers increases the sensitivi-ty of the procedures but also requires abetter quality of starting materials, in-cluding genomic DNA. Most manufac-

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Figure 2. The crude DNA preparations from cotton leaves and pine nee-dles contain potent PCR inhibitors that can be relieved by inclusion of0.1% BSA and 1% PVP. Lane 1, DNA ladder (Invitrogen). Lanes 2 and 3,negative controls. Lanes 4–7, comparisons of CsCl-purified Arabidopsis ge-nomic DNA with crude genomic DNA prepared with our method. Lanes 4and 5 contain 10 ng CsCl-purified Arabidopsis genomic DNA; lanes 6 and 7contain 1 µL crude Arabidopsis DNA. Lanes 8–17 contain 1 µL crude Ara-bidopsis DNA plus 1 µL crude DNA from tobacco, sorghum, cotton, moss,and pine needles, respectively, as shown in the figure. PCR mixtures wereamplified with Arabidopsis marker T4C21. +, inclusion of 0.1% BSA and1% PVP in PCR mixture. -, no BSA or PVP in PCR mixture.

Figure 3. The simple high-throughput DNA extraction method is applic-able to a range of plant species. The crude DNA preparations from Ara-bidopsis, tobacco, sorghum, cotton, star mosses, and pine needles were am-plified with species-specific primers. Arabidopsis was amplified with markerT4C21. Transgenic tobacco was amplified with primers designed from theGFP transgene sequence. Sorghum DNA was amplified with primers de-signed from a drought-inducible expressed sequence tag (BG933199.1). Cot-ton was amplified with primers designed from cellulose synthase A1(U58283.1). Moss was amplified with primers designed from rehydrin geneTr288 (AF275946). Pine was amplified with primers designed from trans-cinnamate-4-hydroxylase gene (AF096998.1). +, inclusion of 0.1% BSA and1% PVP in PCR mixture. -, no BSA or PVP in PCR mixture.

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turers recommend the use of highly pu-rified genomic DNA requiring eitherthe use of an expensive purification kitor a lengthy and complicated procedure(14,15). To increase the usefulness ofour simple method, we determined ifthe genomic DNA in our extractswould be compatible with the use offluorescence-labeled primers for PCRfragment analysis. The forward primerfor the Arabidopsis marker T4C21 waslabeled with either 6-FAM (blue),HEX (green), or NED (yellow).These primers were used to amplify themarker from Arabidopsis ecotypes Co-lumbia, Landsberg, and Wassilewskija,respectively. The resulting fragmentswere analyzed by the use of an ABI3100 Genetic Analyzer (Applied Bio-systems, Foster City, CA, USA) (Fig-ure 4). The genomic DNA preparedwith our simple high-throughput meth-od served as an efficient substrate foramplification with fluorescence-la-beled primers. The DNA samples wereso efficient as PCR templates that wewere able to use the fluorescence-la-beled primer at one-tenth of the recom-

mended concentration. This gave us thebenefit of an additional cost reductionfor fragment analysis using fluores-cence-labeled primers.

DISCUSSION

We have developed a simple high-throughput DNA extraction methodthat can be effectively used for a widerange of plant species, including planttissues that have proved difficult in thepast, such as pine needles. We havemade two significant improvements toexisting DNA extraction methods(7,10). First, we have included 2%Tween 20 in the DNA extractionbuffer, eliminating the need for tissuemaceration. This substantially increas-es the efficiency of processing plantsamples; more importantly, it greatlyreduces the chances of cross contami-nation of samples normally associatedwith high-throughput processes in 96-well plates. Second, we have includeda combination of BSA and PVP in thePCR mixture, alleviating the inhibi-

tion of Taq DNA polymerase associat-ed with unknown components presentin several crude DNA preparationsand thus increasing the utility of oursimple method.

Although commercially designedhigh-throughput DNA extractionmethods are available (16), thesemethods require expensive roboticsand reagents, preventing their use bymany small laboratories. Simple DNAextraction methods that do not requiretissue grinding are also commerciallyavailable (Sigma Extract-N-Amp se-ries). These methods may also beamenable to adaptation to high-throughput platform; however, theminimum cost of $1.50/sample is gen-erally prohibitive for large samplenumbers. The method we describe issimple, adaptable for high throughput,versatile, and cost effective. Our meth-od allows a single researcher to pro-cess several thousand plant tissue sam-ples to the point of PCR setup in oneday without the aid of robotic tech-niques. The estimated cost of ourmethod is less than $0.05/sample. Our

Figure 4. The crude DNA prepared with the simple method is compatible with fluorescence-labeled primers. The crude DNA from Arabidopsis ecotypeColumbia, Landsberg, and Wassilewskija were amplified separately with Arabidopsis marker T4C21 labeled with 6-FAM (blue), HEX (green), and NED (yel-low, shown as black). The PCR products were pooled and separated with ABI 3100 Genetic Analyzer with a 36-cm capillary column and a 45-min run.

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procedure will enable many small lab-oratories to make use of high-through-put technologies currently denied thembecause of high cost. The procedurewill help large laboratories to increaseefficiency and reduce cost.

The amount of tissue needed forthis method is very small, with a leafdisc approximately 6 mm in diameterproviding sufficient genomic DNA for100 20-µL reactions. We have ob-served that successful DNA extractionis possible with a wide range of samplesizes and tissue types. In an Arabidop-sis mapping project, we routinely takewhatever tissue types are the most con-venient to collect, such as youngleaves, cauline leaves, flower buds,small seedlings, etc. However, for cot-ton and pine tissues that contain potentPCR inhibitors, smaller and youngertissues tend to be more successful thanmore mature tissues.

The DNA isolated with our methodis very stable. We have observed thatthe DNA can be stored at 4°C formore than a month and at -20°C forover three months without any appar-

ent effect on the yield and specificityof PCR.

Map-based cloning, large-scaleidentification of tagged mutants,TILLING, and marker-assisted plantbreeding will continue to play keyroles in functional genomics. All ofthese technologies require PCR ampli-fication of a large number of individ-ual or pooled samples. Processinglarge quantities of samples to extractgenomic DNA of a quality that allowssatisfactory PCR amplification oftenrepresents a major limiting step in theuse of these technologies. The methodwe describe largely eliminates thisproblem, especially for small laborato-ries that cannot afford robotics or ex-pensive kits. The quality of DNA pre-pared with this method is compatiblewith the use of both unlabeled and flu-orescence-labeled primers for PCR.The procedure is reliable and repro-ducible, typically displaying a successrate of over 95%.

DISCLAIMER

Mention of trade names or commer-cial products in this article is solely forthe purpose of providing specific infor-mation and does not imply recommen-dation or endorsement by the U.S. De-partment of Agriculture.

ACKNOWLEDGMENTS

We thank Jeremy Thomas for histechnical support. We thank CereonGenomics, LLC, for making its DNAmarker database publicly available.

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Received 9 December 2002; accepted6 February 2003.

Address correspondence to:Dr. Zhanguo XinUSDA-ARSPlant Stress and Germplasm Development

Laboratory3810 4th Street Lubbock, TX 79415, USAe-mail: zxin@lbk.ars.usda.gov