HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Flagel, L. Articles by Matthews, P. D. Search for Related Content PubMed Articles by Flagel, L. Articles by Matthews, P. D. Agricola Articles by Flagel, L. Articles by Matthews, P. D. Related Collections Crop Genetics Soybean

GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY NOTE

Inexpensive, High Throughput Microplate Format for Plant Nucleic Acid Extraction

Suitable for Multiplex Southern Analyses of Transgenes

^a Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, St. Paul, MN 55108
^b BASF Plant Science, 26 Davis Drive, Research Triangle Park, NC 27709
^c Crop Improvement, S.S. Steiner, Inc., 655 Madison Ave, New York, NY 10021

^* Corresponding author (pmatthews{at}hopsteiner.com)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
To achieve high throughput analysis of genomic DNA from small amounts of plant tissue, a standard nucleic acid extraction was formatted to microplates by linking a novel series of interlocking plates. The protocol integrates steel pellet tissue homogenization and unique liquid handling and phase separation techniques with minimal special equipment. The method was validated for difficult plant DNAs, such as hop (Humulus lupulus L.), soybean [Glycine max (L.) Merr.], and tobacco (Nicotiana tabacum L.), with agarose gel separations of restriction fragments and Southern blot analysis of transgene integrants in soybean. The range of success rates for Southern blots was 73 to 92% per sample per plate (n = 2016 samples). Variation in absolute yield was quantified by PicoGreen microplate flourimetry. The variation in yield among samples per plate was small enough to obviate individual sample concentration adjustment before Southern analysis. Average absolute yield for the barley (Hordeum vulgare L.) mapping population based on flourimetry was 6.8 µg ± 0.85 SE, n = 96; providing enough pure, stable DNA for many individual polymerase chain reaction (PCR) reactions. Intersample cross-contamination and suitability for PCR analysis were validated by simple sequence repeat (SSR) display of a previously characterized barley mapping population. Cost calculation from a materials detail was U.S. $0.17 per sample extraction. Although the implementation requires some skill and practice, we find it to be a robust, adaptable, and versatile alternative to expensive commercial kits.

Abbreviations: CTAB, cetyltrimethylammonium bromide • PCR, polymerase chain reaction • SSR, simple sequence repeat

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
EXTRACTION AND ANALYSIS of plant DNA is often the bottleneck in the production and analysis of transgenic plants (Lin et al., 2001). In addition, PCR analysis is not appropriate for detection of stable integration of genes, especially in Agrobacterium-mediated transformation, and invariably provides less information than restriction analyses and Southern blots. Southern detection from small amounts of tissue is desirable during the transformation process, for example, in confirmation of transgenic status of calli and shoots. However, most multiplex techniques produce amounts and quality of DNA insufficient for Southern blot analysis and only suitable for PCR (Dilworth and Frey, 2000; Karakousis and Langridge, 2003; Lange et al., 1998; Mace et al., 2003; Shepard et al., 2002; Xin et al., 2003).

Inexpensive extraction may also improve the cost-effectiveness margin of molecular marker-assisted breeding over convention breeding (Moreau et al., 2000; Hill-Ambroz et al., 2002; Willcox et al., 2002). RFLP markers are less often used nowadays, although they offer many advantages, due in part to the cost of extracting sufficient quantities of DNA. Many passive ("soak and hope") extraction techniques have been developed to extract small amounts of crude DNA for PCR-based analyses, but difficulties may occur in subsequent modification by restriction endonucleases and ligase for amplified fragment length polymorphism production (Lin et al., 2001). The amount and quality produced in such crude DNA preps limits the number of downstream applications to only a few (Lin et al., 2001) and limits the capability to store the DNA long-term. Glass fiber filter techniques (Lange et al., 1998) have been adapted for plant DNA, but may not work for some phenolic-ridden plants, produce partially degraded DNA, and furthermore are often expensive.

Our protocol is simply the "tried and true" in a new tube. We used a modified cetyltrimethylammonium bromide (CTAB) extraction buffer (Doyle and Doyle, 1987, 1990). Phenol/chloroform extractions and alcohol precipitations were forced into the microplate format. The use of the 96-well uniplate required the congregation of a few familiar tricks and little special equipment, namely: (i) freezing and powdering small tissue samples with a single steel pellet (BB) in a 96-well block with a paint shaker, (ii) application of buffer with a home-made 96-well funnel and standard microplate centrifuge, (iii) facilitation of separations of phenol/chloroform extractions with phase-lock gel, which allows decanting of all 96 samples simultaneously, (iv) aiding fluid transfers by a series of snuggly interdigitating 96-well plates, and (v) centrifugation of alcohol precipitations for extended times into conical microplate wells.

The protocol used microtiter-format gel electrophoresis equipment for downstream applications. A number of scale options are presented for genomic DNA yields ranging from 1 to 20 µg. Low variability in yield among sample wells, likely due to the relative thoroughness and evenness of mechanical grinding (Michaels and Amasino, 2001; Hill-Ambroz et al., 2002), makes DNA concentration measurement and adjustment unnecessary for Southern blotting. Examples of application to difficult plants such as hop, tobacco, and soybean are given, including representative data from a large-scale analyses by Southern blotting of soybean primary transformants and subsequent sexual segregations of transgenes (Olhoft et al., 2003; Olhoft et al., 2004). The procedure was also validated for use in preparation of PCR template by application of SSR amplification to a previously characterized barley mapping population.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Barley progeny from a cross of Atahualpa (a cultivated two-row variety from South America) and M81 (a University of Minnesota elite six-row variety) were grown in the greenhouse. Kiln-dried hop (‘Galena’) cones and processed hop pellets were from S.S. Steiner, Inc. Transgenic soybean (‘Bert’) families (Olhoft et al., 2003) were grown in the greenhouse. Tobacco (‘Samson’) was grown in the greenhouse. All extractions were done from immature (not fully expanded) leaves that were harvested directly to microplates in the greenhouse or from detached leaves stored at 4°C overnight, except in the case of hop, where the tissue is a kiln-dried processed agricultural product: namely, the trichomatous glands and the bracts and bracteoles of the female hop flower. All chemicals were purchased from the Sigma Chemical Company (St. Louis, MO).

Photographs of the procedure steps and a short, printable protocol reside at http://bioplasticscollaborative.coafes.umn.edu/uniplate_dna_extraction.htm.

Uniform leaf discs were made with a 18-mm cork borer, except in the case of barley, where 2-cm cross sections of leaf blades were cut with scissors, and of hop, where individual whole bracts or processed product (pellets) were used. Samples weighed about 0.2 g. Leaf samples were rolled up and placed into individual 1-mL wells of a riplate deep-well microplate (Continental Lab Products, San Diego, CA). Alternatively, larger tissue samples (1 g) were processed starting with a Megatiter plate (Continental Lab Products, San Diego, CA). The plate containing samples was then frozen in a liquid nitrogen bath and immediately stored frozen at –80°C. (Leaf discs may be harvested directly to plates cooled on dry ice or liquid nitrogen.) While still frozen, one zinc-plated steel pellet (BB, Daisy, Rogers, MN) was placed in each well and pushed down with a screwdriver to crush the tissue. The steel pellet must have a free path for motion within the tube of about 1 cm. The plate was covered with strip caps (Continental Lab Products, San Diego, CA) hammered on with a rubber hammer, and then shaken in a Model 5400 paint can mixer (Red Devil, Brooklyn Park, MN). Commercially produced beadmills are available, but are more expensive. The samples were shaken until a fine powder was produced, usually three cycles of 30 s shaking with refreezing in nitrogen and reorienting of the plate within the shaker clamp between shake cycles. The grindates were allowed to warm slowly by placing the sample block at –20°C. This avoided explosive air pressure changes, which may dislodge the caps.

To facilitate timely, efficient, and quick dispensing of extraction buffer to each frozen grindate, microplate funnels were created and used to apply buffer simultaneously to plates by centrifugation. Buffer loading plates are prepared in advance and held at 65°C until use. Holes were punched with a 21-gauge hypodermic needle into the bottoms of the wells of a 300-µL chimney stack 96-well microplate (Continental Lab Products, San Diego, CA). The pinholes were sealed by pushing the funnel onto a microplate-sized piece of Parafilm sealing tape (American National Can, Chicago, IL). The funnel plate was filled with 300 µL of preheated (65°C) extraction buffer (100 mM Tris HCL pH 6.0, 25 mM EDTA disodium salt, 1.5 M NaCl, 2.5% CTAB, 2% polyvinyl pyrrolidone MW 40000, 0.5% N-lauroylsarcosine, 0.5% diethylthiocarbamate) with a 12 channel micropipettor and stored in an oven until use. The Parafilm was removed and the hot funnel plate quickly placed on top of the sample plate that had been placed in a Beckmann TJ-6 centrifuge with a TH-4 rotor and microplate carriers at 20°C (Beckmann Instruments Inc., Palo Alto, CA). A 5-min centrifugation step infused the buffer into the grindate. After buffer infusion, the plate was capped and gently hand-shaken several times during incubation at 65°C for 5 min. The block was then shaken standing on its longest side for an additional 20 min at 100 rpm on an orbital platform shaker at room temperature. At this point, one option is removal of the caps and occlusion of the wells with phase lock gel, as described below. The plate was then centrifuged for 10 min at 3200 rpm. Centrifugation yielded a tissue pellet with the BB below and the aqueous extract above. Transfer of 200 µL of the extract was achieved with a multichannel pipettor or by decanting to a fresh riplate, that has been previously filled with 100 µL per well of phase lock gel (Brinkmann Eppendorf, Westbury, NY). This phase lock block was prepared in advance by spreading the gel-like butter across the top of the plate with a tongue depressor and centrifuging the gel to the bottom of the wells. Four hundred µL of phenol/chloroform/isoamyl alcohol 25:24:1 was placed in each well with the extract and plates were capped and shaken for 20 min on an orbital platform. Plates were centrifuged. Subsequently, the aqueous phase, which remained above the gel layer, was decanted into a chimney stack plate by interlocking the stacks with the wells and flipping the interdigitated plates upside down. The extract was then transferred into a fresh phase lock block for extraction with 200 µL of chloroform. The two-step transfer allowed retention of the original cell addresses, but, if not done carefully, the process can reverse the well addresses. After the organic extraction was completed, the aqueous phase was decanted to chimney stack plate by flipping as described before. The well addresses were now reversed. The reversal was usually corrected in the first pipetting step of the downstream process. Nucleic acids were precipitated by the addition of 180 µL of isopropanol, followed by capping, mixing by inversion, and incubation at –20°C. Extracts may be stored overnight at this point. Nucleic acids were pelleted in the chimney stack plates by centrifugation at 3200 rpm for 1 h. After centrifugation, the fluid phase was poured out by careful inversion of the plate and blotting onto absorbent paper. The pellets were washed with 200 µL of cold 70% ethanol and centrifuged for at least 20 min. The ethanol wash was decanted. Remaining ethanol can be evaporated away in a 37°C incubator or centrifugal vacuum dryer. Pellets were dissolved by overnight incubation at 4°C in 22 µL of sterile water containing 0.02% v/v RNase (10 mg mL^–1).

A restriction digestion mix containing 3 µL of 10 x BSA, 3 µL of 10 x restriction buffer (as supplied by the manufacturer), and 2 µL of restriction endonuclease (20 units) was prepared. Eight microliters of this mix was added to each 22 µL of DNA solution, and the reaction was incubated for 4 h at 37°C. Five microliters from each well of one row was selected for minigel analyses of restriction fragmentation process. If the digestion was successful, the samples were evaporated to 15 µL for analysis on a 100-well microtiter format gel. Samples are loaded in a gel with a multichannel pipette. After electrophoresis, DNA was transferred to Immobilon-Ny⁺ (Millipore, Bedford, MA) membranes according to the manufacturer's protocol.

All other separation and detection protocols, including radiolabeling and Southern hybridization, PCR amplification, and polyacrylamide gel electrophoresis separation of SSRs, were performed with standard protocols and with commercially available kits. DNA imaging was performed with a Gel Doc system (Biorad, Hercules, CA), ß-radiation imaging with a Storm 840 phosphoimager (Molecular Dynamics, Sunnyvale, CA); Picogreen (Molecular Probes, Eugene, OR) DNA quantification was performed with a Benchmark Plus microplate spectrophotometer (Biorad, Hercules, CA). Simple sequence repeats were visualized by silver staining. Images were processed using Photoshop 5.5 (Adobe, San Jose, CA).

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Our method was in part devised to deal with the need for microgram quantities of highly purified DNA from common agricultural and laboratory model plants, including tobacco, soybean and hop. Previously, we had been experiencing difficulty in producing good Southern blots with DNA from these phenolic-containing plants. The complicated buffer system and low pH were chosen to inhibit oxidation and polymerization of polyphenols that often co-purify with DNA (Csaikl et al., 1998). Figure 1 demonstrates that we were able to obtain high molecular weight DNA (modal size = approximately 30 kb) from hop, barley, soybean, and tobacco. Degraded DNA suitable for PCR (modal size = approximately 6 kb) was also extracted from commercially pelletized and thus thermally damaged hop. All high molecular weight DNA extracts were susceptible to complete nuclease digestion (data not shown), as demonstrated for tobacco and soybean DNA in Southern blotting and detection systems. Freeze-dried tissues were not used here, as the drying process leads to partially degraded DNA, which is not suitable for detection of restriction fragment length polymorphisms.

View larger version (107K): [in this window] [in a new window]   Fig. 1. Extracted DNA from various plants is of high molecular weight and susceptible to endonuclease digestion. Agarose gels stained with ethidium bromide: (A) barley undigested, close-up; (B) hop, undigested, close-up; (C) hop pellets, degraded in food processing, close-up; (D) tobacco, complete set of 96 extractions separated on a two-tiered gel, HindIII; (E) soybean, complete set of 96 on a two-tiered gel, HindIII. Molecular weight markers: 1 kb ladder (Invitrogen, Carlsbad, CA), except B and D, Hind III markers.

As part of a study of transgene segregation, the method was applied to 2016 soybean plants (Olhoft et al., 2004). Figure 2 shows a representative Southern blot detection of independently transformed soybean lines. Segregation of transgenes among progenies of primary transformants were detected and scored on more than 10 such blots (Fig. 2), each with restriction digests of genomic DNA from 192 individual extractions. The success rate per individual transgenic progeny was visually assessed by gel electrophoresis, ethidum bromide staining, and Southern hybridization to a transgene probe. The most frequent (5.6%) failure was loss of sample, which may occur to whole rows, followed by lack of susceptibility to endonuclease digestion (2.4%), and degradation of high molecular weight DNA (0.25%). The rate of sample loss depends on the care and skill of the experimenter, while the causes of the latter failures are unknown. The overall success rate for Southern detection was 92%.

View larger version (133K): [in this window] [in a new window]   Fig. 2. Example of soybean Southern detection of primary transformants probed with the Hpt (hygromycin phosphotransferase) gene.

The method offers several advantages over other microplate protocols. It is inexpensive and needs little special equipment. Many of the components, such as riplates and chimney stack plates, can be reused to further lower the cost. Reuse of plates requires washing and baking or autoclaving of plates. Commercial DNA decontamination treatments are also available. Estimates for cost of chemicals and plasticware were from US $0.17 (with recycling of plasticware) to US $0.27 per individual extraction. Importantly, some laborious and error-prone activities are obviated. Multichannel pipetting transfers are replaced by simultaneous 96-well decants. Transfer of labels was eliminated by using the microplate format. Therefore, one person can produce 384 extractions in one 10-h day, including tissue sampling. The protocol is laborious and requires skill and attention to detail. More often, the method was broken into legs by storage of tissue samples at –80°C until extraction and overnight precipitations were maintained at –20°C, as indicated.

We show that the method is versatile for a number of downstream applications. While the method was devised and demonstrated for cost-efficient Southern detection of transgene loci, it also produces a template suitable for PCRs. The yield of DNA allows for thousands of PCRs per extract, and the purity of the DNA should allow good shelf life (not tested). Therefore, this protocol may be more efficient in some molecular mapping efforts, as it reduces the number of tissue resampling efforts compared with resin or filter-based extraction techniques, which have much lower absolute yields and produce sheered DNA. Furthermore, the DNA is not matrix bound, facilitating manipulations in strategies requiring pooling of individual extracts (Lange et al., 1998).

Many other chemical extractions may be more economical with this combination of efficient multiplex homogenization, gel-facilitated phase separations, and simple decants. The extraction buffer described here may be substituted with ones particular to a specific plant, laboratory, or downstream application, such as selective purification of RNA. The protocol might be modified for a large variety of metabolites other than nucleic acids. Although more laborious, our approach could be much less expensive than commercially available, automated solid phase extraction techniques for nucleic acids and other metabolites and, therefore, within the capacity of numerous laboratories (Mace et al., 2003).

	ACKNOWLEDGMENTS

 
We would like to thank University of Minnesota Department of Plant Genetics and Agronomy reviewers and Dr. Miguel Cervantes-Cervantes and Christina Murillo for critical review of this manuscript.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
This research was supported in part by the Minnesota Soybean Research and Promotion Council and by S.S. Steiner, Inc.

Received for publication November 10, 2004.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

• Csaikl, U.M., H. Bastian, R. Brettschneider, S. Gauch, A. Meir, M. Schauerte, F. Scholz, C. Sperisen, B. Vornam, and B. Ziegenhagen. 1998. Comparative analysis of different DNA extraction protocols: A fast, universal maxi-preparation of high quality plant DNA for genetic evaluation and phylogenetic studies. Plant Mol. Biol. Rep. 16:69–86.
• Dilworth, E., and J.E. Frey. 2000. A rapid method for high throughput DNA extraction from plant material for PCR amplification. Plant Mol. Biol. Rep. 18:61–64.[ISI]
• Doyle, J.J., and J.L. Doyle. 1987. A rapid DNA isolation procedure from small quantities of fresh leaf tissue. Phytochem. Bull. 19:11–15.
• Doyle, J.J., and J.L. Doyle. 1990. Isolation of plant DNA for fresh tissue. Focus 12:13–15.
• Hill-Ambroz, K.L., G.L. Brown-Guedira, and J.P. Fellers. 2002. Modified rapid DNA extraction protocol for high throughput microsatellite analysis in wheat. Crop Sci. 42:2088–2091.[Abstract/Free Full Text]
• Karakousis, A., and P. Langridge. 2003. A high-throughput plant DNA extraction method for marker analysis. Plant Mol. Biol. Rep. 21:95a–95f.
• Lange, D.A., S. Penuela, R.L. Denny, J. Mudge, V.C. Concibido, J.H. Orf, and N.D. Young. 1998. A plant DNA extraction protocol suitable for polymerase chain reaction based marker-assisted selection. Crop Sci. 38:217–220.[Abstract/Free Full Text]
• Lin, R.-C., Z.-S. Ding, L.-B. Li, and T.-Y. Kuang. 2001. A rapid and efficient DNA minipreparation suitable for screening transgenic plants. Plant Mol. Biol. Rep. 19:379a–379e.
• Mace, E.S., H.K. Buhariwalla, and J.H. Crouch. 2003. A high-throughput DNA extraction protocol for tropical molecular breeding programs. Plant Mol. Biol. Rep. 21:459a–459h.
• Michaels, S.D., and R.M. Amasino. 2001. High throughput isolation of DNA and RNA in 96-well format using a paint shaker. Plant Mol. Biol. Rep. 19:227–233.
• Moreau, L., S. Lemarié, A. Charcosset, and A. Gallais. 2000. Economic efficiency of one cycle of marker-assisted selection. Crop Sci. 40:329–337.[Abstract/Free Full Text]
• Olhoft, P.M., L.E. Flagel, C.M. Donovan, and D.A. Somers. 2003. Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216:723–735.[ISI][Medline]
• Olhoft, P.M., L.E. Flagel, and D.A. Somers. 2004. T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method. Plant Biotechnol. J. 2:289–300.[CrossRef][Medline]
• Shepard, M., M. Cross, R.L. Stokoe, L.J. Scott, and M.E. Jones. 2002. High-throughput DNA extraction from forest trees. Plant Mol. Biol. Rep. 20:425a–425j.
• Willcox, M.C., M.M. Khairallah, D. Bergvinson, J. Crossa, J.A. Deutsch, G.O. Edmeades, D. González-de-León, C. Jiang, D.C. Jewell, J.A. Mihm, W.P. Williams, and D. Hoisington. 2002. Comparison of phenotypic and marker-assisted selection for quantitative traits in sweet corn. Crop Sci. 42:1516–1528.[Abstract/Free Full Text]
• Xin, Z., J.P. Velten, M.J. Oliver, and J.J. Burke. 2003. High-throughput DNA extraction method suitable for PCR. Biotechniques 34:820–826.[ISI][Medline]

This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Flagel, L. Articles by Matthews, P. D. Search for Related Content PubMed Articles by Flagel, L. Articles by Matthews, P. D. Agricola Articles by Flagel, L. Articles by Matthews, P. D. Related Collections Crop Genetics Soybean