MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY An Inexpensive, Simple Protocol for DNA Isolation from Blood for High-Throughput

Genotyping by Polymerase Chain Reaction or Restriction Endonuclease Digestion

S. M. Bailes,* J. J. Devers, J. D. Kirby,1 and D. D. Rhoads2
*Missouri State Highway Patrol Crime Laboratory, Jefferson City 65101; Program in Cell and Molecular Biology; Department of Poultry Science; and Department of Biological Sciences, University of Arkansas, Fayetteville 72701 ABSTRACT We describe simple, inexpensive, and reliable methods for isolating DNA from avian blood, semen, or feather pulp. The procedures are readily applicable to high-throughput 96-well plate isolation for genotype analysis of chicken DNA based on restriction endonuclease digestion or PCR. Isolation cost is primarily the cost of a deep-well assay block and a few pipet tips; current price is less than $0.10 per sample, providing a signicant cost advantage over commercial kits. The procedure employs inexpensive, nonhazardous reagents and yields intact, double-stranded DNA from as little as 2 to 10 L of avian blood, suitable for RFLP analysis or hundreds of PCR amplications. We compared our method to published procedures for alkaline extraction from feather pulp and found our method to be more reliable with the advantage of isolating intact DNA sequences that can be easily quantied. With minor modications, the method can isolate DNA for PCR genotyping from mammalian whole blood.

Key words: avian blood, DNA isolation, polymerase chain reaction, restriction fragment length polymorphism 2007 Poultry Science 86:102106

INTRODUCTION
High-throughput PCR-based genotyping requires simple methods for DNA purication. Use of DNA isolation kits in 96-well format to isolate sufcient DNA for 50 or more PCR amplications can be expensive, with retail prices greater than 10-fold more costly than methods employing standard reagents (e.g., QIAamp, Qiagen, Valencia, CA; or Wizard SV96, Promega Corp., Madison, WI). Cost per sample can make highthroughput genotyping cost prohibitive for application to agricultural species, especially poultry. Less expensive commercial kits (e.g., Extract-N-Amp, Sigma-Aldrich, St. Louis, MO) isolate DNA sufcient for only a few PCR reactions, and are therefore not suitable for genomic analyses for many loci in genomic scans. Most methods for isolation of highly puried, intact DNA from blood use organic extractions (hazardous chemicals), ethanol precipitation with high-speed centrifugation (not amenable to 96-well assay blocks), and numerous individual steps (labor intensive). An inexpensive simple method that uses 30-min NaOH treatments has been used to extract DNA from blood (Rudbeck and Dissing, 1998) or feathers (Malago et al., 2002). These

techniques isolate sufcient DNA for 50 to 100 PCR reactions. However, the alkaline treatment also denatures the DNA, leaving it unsuitable for quantication by sensitive intercalating-dye-uorescence or analysis by restriction endonuclease digestion. More laborious methods that isolate double-stranded DNA introduce chemicals (EDTA, SDS, NaI) that must be removed through organic extraction or ethanol precipitation before enzymatic treatment (e.g., restriction endonuclease digestion or PCR) (Miller et al., 1988; Grimberg et al., 1989; Loparev et al., 1991; Yokota et al., 1998). An inexpensive method has been described whereby nuclei are puried followed by proteinase K digestion (Ding, 1992). We have adapted this procedure to a 96-well format and replaced the proteinase K treatment to develop a protocol for isolation of double-stranded DNA sufcient for hundreds of genotype determinations. The cost is as little as $5 per 96well plate. The procedure produces DNA suitable for sensitive quantication by Hoechst 33258 dye uorescence, PCR analysis, or restriction digestion for Southern blot or RFLP analysis. We have applied this procedure to high-throughput PCR screening of avian blood samples, and for isolation from feather pulp. We have also demonstrated that our procedure can be easily modied for use on mammalian whole blood.

2007 Poultry Science Association Inc. Received April 4, 2006. Accepted August 2, 2006. 1 Present address: Department of Animal and Range Sciences, South Dakota State University, Brookings 57007. 2 Corresponding author: drhoads@uark.edu

MATERIALS AND METHODS DNA Isolation

Whole blood samples were collected via Vacutainer (Becton Dickinson, Franklin Lakes, NJ) blood collection 102

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1

tubes containing EDTA and stored frozen in microfuge tubes or 96-well assay blocks, at 20C. Semen samples were collected by manual manipulation of males, transferred to 96-well assay blocks, and frozen at 20C. The DNA was isolated in conical 0.5-mL polypropylene 96well assay blocks (Corning, Inc., Corning, NY). Wells were preloaded with 0.2 to 0.5 mL of cold STM buffer (64 mM sucrose, 20 mM Tris Cl, pH 7.5, 10 mM MgCl2, and 0.5% Triton X-100) and held on ice. For highthroughput screening of frozen chicken blood, 2 to 3 L was added to each well with a sterile toothpick. The sterile at toothpick was placed about 3 to 5 mm into the partially frozen blood and then transferred to a well of the assay block containing STM. The toothpicks were discarded once all wells were loaded. Larger volumes of blood (10 to 30 L) were triturated into 0.5 mL of STM buffer. After addition of blood, nuclei were pelleted at 1,000 g for 5 min in a centrifuge equipped to spin microplates. The assay block was inverted to decant the supernatant and allowed to drain briey on a paper towel. Nuclear pellets were resuspended by trituration with a multichannel pipettor to disperse the pellet into 200 L of Tris-EDTA-NaCl + pronase solution (TEN+pronase; 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 10 mM NaCl, 100 g/mL of pronase; Sigma-Aldrich, St. Louis, MO). Precut sealing tape (Corning Inc. Life Sciences, Acton, MA) was used to seal the assay blocks, which were then incubated for at least 1 h (when blood volumes exceeded 10 L, we extended the pronase treatment to 2 h to ensure adequate digestion) with shaking at 37C in a bacteriological incubator, then at 65C in a water bath for 10 to 30 min to inactivate the pronase. For semen samples, lysis was found to be unnecessary. Semen (5 to 10 L) was triturated into 0.2 mL of TEN+pronase, digested for 1 h at 37C, and then inactivated for 10 min at 65C. Feather pulp samples were treated in the same way as semen samples: 2 pieces comprising approximately 4 to 5 mm from the tips of tail or wing feathers were incubated for 1 h in 0.2 mL of TEN+pronase and then inactivated for 10 min at 65C. For isolation from fresh bovine blood, 100 L of blood was lysed in 800 L of STM. The rst nuclear pellet was suspended in a new aliquot of STM buffer, and then nuclei were re-pelleted. The nuclear pellet was then suspended in 200 L of TEN+pronase, digested for 1 h at 37C, and then inactivated at 65C for 10 min. Isolation of DNA by alkaline extraction from feather pulp was as described (Rudbeck and Dissing, 1998; Malago et al., 2002). Feather tips (approximately 5 mm as 2 pieces) were incubated in 50 L of 100 mM NaOH for 1 h at 37C and then neutralized with 240 L of 40 mM TrisCl, pH 7.5. Isolated DNA samples were stored frozen at 20C. Final DNA quantication was by Hoechst 33258 uorescence measured in a uorometer (model TKO, Hoefer Scientic Instruments, San Francisco, CA) according to manufacturers protocols.

Table 1. Yield of DNA from a variety of sources

Yield (g) Source Chicken blood, frozen Chicken blood, fresh Volume (L) Toothpick 2 5 10 20 30 5 10 200 n 25 2 2 2 2 2 9 6 20 3 Range 1.6 to 88 25 to 30 40 to 41 40 to 60 100 to 112 125 to 147 15 to 40 30 to 40 2 to 10 3 to 5 Average SD 20 22

Chicken semen, frozen Chicken feather tip Bovine blood, frozen

1

26 8 34 4

The indicated volumes of sample from the indicated source were processed according to our described procedures (Materials and Methods). For each source and volume, the number of samples (n) is indicated as well as the range of DNA yield. Where 3 or more samples were processed, the average yield and standard deviation (SD) were calculated.

Restriction Endonuclease Digestion

Genomic DNA samples (3 to 4 g) were digested in 50-L reactions using buffers and conditions recommended by the enzyme supplier (Promega Corp., Madison, WI). After digestion, DNA samples were resolved in 0.7 or 1.5% agarose gels in 0.5 Tris borate EDTA (50 mM Tris borate, pH 8.3, 1 mM Na2EDTA), stained with ethidium bromide, and detected by uorescence (532 nm excitation, 610 nm emission) with a Typhoon 9600 scanner (GE Health Care, Piscataway, NY). Selected gels were further analyzed by Southern blot hybridization (Nakamichi et al., 1983).

PCR Genotyping
Microsatellite genotype determination used uorescent-labeled primers (custom synthesis, MWG Biotech, High Point, NC). We developed primer ADR001 (5HEX-gcttcgactatctagaatg-3, 5-gctaaaatataaaatgcagg-3) as a specic marker for the estrogen receptor (ER gene on chicken chromosome 3 (unpublished); KS001 (5-FAM-gatcattgctgcaaaatgga-3, 5-gaaggtgactcagattagg-3) is specic for a locus on chicken chromosome 17 (unpublished); and LEI0217 (5- HEX-gatgactgagagaaataacttg-3, 5-aaattactgaggcacaggag-3) and UMA1.070 (5- HEX-aagcttttaaaccaatctga-3, 5-tcctgcatgtgccctttgta-3) derive from chicken chromosome 1 (Schmid et al., 2000). The DNA samples (1 to 2 L) were amplied in 20-L PCR reactions in 200-L 96-well PCR plates (VWR International, Bristol, CT) in a thermocycler (model PTC-100, MJ Research, Watertown, MA). Each PCR reaction contained 1 PCR buffer (50 mM TrisCl, pH 8.3, 1 mM MgCl2, 30 mg/mL of BSA), 0.2 mM dNTP, 0.2 M of microsatellite primers, and 5 U of Taq polymerase. Reactions were either overlaid with 20 to 50 L of mineral oil, or sealed with tape. The PCR conditions were 90C for 2 min, 45 cycles of 90C for 30 s, 45C for 1 min, 72C for 1 min, followed by 72C extension for 7 min. Products were either resolved on 30 40 cm 6%

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Figure 1. Comparison of DNA restrictability: Southern blot of HindIII digested DNA samples (2 g/lane) probed with a cDNA for estrogen receptor . Lanes 1 to 3: DNA samples prepared by our method; lanes 4 to 5: DNA samples puried by standard methods. Molecular weights based on DNA standards are indicated to the right of the gure. Figure 2. Polymerase chain reaction sexing of DNA samples isolated by alkaline extraction or by pronase digestion. The DNA samples were subjected to PCR using the WXho and Ribo primers, and resolved in a 1.5% agarose gel. Top: 24 DNA samples extracted from feather pulp by the NaOH method from different hens. Bottom: DNA samples extracted from feather pulp by TEN+pronase (Tris-EDTA-NaCl + pronase) digestion from the same hens as in the top panel.

acrylamide denaturing gels, then detected with a Typhoon 9600 scanner, or resolved and detected on an ABI377 sequencer (model ABI 377, Applied Biosystems, Foster City, CA). For the ABI377 sequencer, PCR products were diluted 1:5 with water then 1 L of the dilution was mixed with 3 L of TAMRA-labeled internal line standard (Applied Biosystems) denatured at 100C for 2 min, chilled on ice, and samples (2 L) were loaded and resolved as recommended by the manufacturer. Allele sizes were determined using Genescan software (Applied Biosystems). The PCR analysis for Wxho and Ribo sex determination primers was as described (Mozdziak et al., 2005). Products were resolved on 1.5% agarose gels.

RESULTS AND DISCUSSION

Our DNA isolation technique was developed from a previous method based on cell lysis in hypotonic, nonionic detergent, low-speed centrifugation to pellet nuclei, disruption of nuclei, and partial proteinase K digestion in a low ionic strength buffer (Ding, 1992). We modied this technique for use in 96-well format and used pronase E, a protease that can be denatured at 65C, thus maintaining the native, double-strand form of the DNA. We evaluated the DNA yield from fresh or frozen chicken blood, chicken semen, chicken feather pulp, and bovine blood (Table 1). As can be seen from the data in Table 1, there is a nearly linear relationship between isolated DNA concentration and chicken blood volume using our procedure. The quantity of DNA is sufcient for hundreds of standard PCR genotype analyses (e.g., microsatellite and single nucleotide polymorphism analysis). Our procedure yields intact DNA sufciently pure for restriction endonuclease treatment. We compared DNA isolated from chicken blood by our method to DNA puried using standard SDS lysis, proteinase K diges-

tion, organic extraction, and ethanol precipitation (Grimberg et al., 1989). The DNA aliquots were digested with various enzymes (AluI, BamHI, EcoRI, or HindIII). Electrophoretic patterns from digested DNA were indistinguishable between the DNA isolated by the 2 different methods (data not shown). Southern blot analysis using a cDNA probe to ER (provided by D. Bunik, University of Illinois, Urbana-Champaign) showed no differences between the hybridization patterns (Figure 1). Thus, this method inactivates nonspecic nucleases, introduces no inhibitory compounds, and produces DNA solutions suitable for restriction endonuclease digestion for Southern blot analysis and RFLP genotyping. To evaluate the DNA isolation procedure for highthroughput PCR genotyping, we isolated DNA from 886 frozen blood samples archived for over 1 yr in a frost-free freezer using our toothpick method. Typically, blood samples stored in this manner can be of poor quality (cell lysis) or be difcult to pipet when thawed (clots). After DNA isolation, quantication of 25 random samples showed a variable range of DNA concentrations (Table 1). Given these concentrations, we used 2 L of the DNA solution in 20-L PCR amplications for genotyping at 2 microsatellite loci, ADR001 and KS001. For the 886 archived chicken blood samples, we obtained genotype data for 792 samples for ADR001 and 737 samples for KS001, where 78 samples amplied for ADR001 only, 28 samples amplied for KS001 only, and 68 samples gave no amplication for either locus. The latter are most likely because of low DNA quantities for those samples or pipetting errors. Therefore, using our rapid

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Figure 3. Microsatellite PCR analysis of DNA samples isolated by alkaline extraction or by pronase digestion. The DNA samples were amplied for ADR001 (upper panel) and KS001 (lower panel). Lanes 1 to 10: DNA extracted from feather pulp by NaOH treatment; lane 11: DNA puried from blood by standard methods; lanes 12 to 21: DNA extracted from feather pulp by TEN+pronase (Tris-EDTA-NaCl + pronase) digestion; lanes 22 and 23: no input DNA; lane M: molecular weight markers. Lengths (bp) of marker bands are indicated to the right.

DNA isolation method, our PCR success rate was 818 out of 886 or 92.3%, based on the ability to genotype at least 1 locus for each DNA sample. We have used DNA extracted by our method from semen for hundreds of microsatellite PCR genotype determinations (data not shown). We compared DNA isolated by our method from single feathers to DNA extracted using alkaline treatment (Rudbeck and Dissing, 1998; Malago et al., 2002). Figure 2 shows PCR data comparing these DNA samples when amplied with WXho and Ribo sexing primers. Only 9 of the 24 DNA samples isolated by NaOH extraction yielded adequate amplication, whereas all 24 of the samples isolated by TEN+pronase digestion amplied, with 1 DNA sample producing only weak amplication, and 2 amplifying only the W-specic fragment. We also tested these DNA samples for microsatellite genotyping with ADR001 and KS001 and found that 9 of 10 TEN+pronase DNA samples amplied for both loci whereas zero of 10 NaOHextracted DNA samples amplied for either locus (Figure 3). We tested these same DNA samples for 2 additional microsatellite loci (LEI0217 and UMA1.070) and produced amplications for 3 of 10 NaOH-extracted DNA samples vs. 10 of 10 TEN+pronase-extracted DNA samples (data not shown). Therefore, in our experience, the TEN+pronase digestion is much more reliable and produces DNA more amenable to PCR genotyping. We adapted the procedure to work with mammalian blood samples. Based on the low frequency of nucleated cells in mammalian whole blood and greater starting sample volumes, we found that 2 successive STM treatments were required to reduce contaminating proteins and avoid coagulated proteins upon enzyme denaturation at 65C. When we used 2 successive centrifugation steps we obtained nal DNA that could be successfully amplied in PCR using primers to bovine HSP70 (provided by C. Rosenkrans, University of Arkansas, Fayetteville; data not shown). This simple, and inexpensive method for isolation of DNA from a variety of sources will facilitate the rapid,

high-throughput PCR or RFLP analysis of genotypes in avian and other species in situations where overall costs must be minimized.

ACKNOWLEDGMENTS
This work was partially supported by grants to JDK and DDR from the Arkansas Biosciences Institute, and from Cobb-Vantress Inc., Siloam Springs, AR. Katrina Collins, and Laura Thorne provided valuable input and technical assistance. Special thanks to Bobbi Okimoto for assistance and training in the UA DNA Core Laboratory.

REFERENCES
Ding, Y. 1992. DNA preparation from trace forensic biological evidences. Pages 371375 in Handbook of PCR Gene Amplication Experiments. P. Zhu, ed. Chin. Sci. Technol. Press, Beijing, China. Grimberg, J., S. Nawoschik, L. Belluscio, R. McKee, A. Turck, and A. Eisenberg. 1989. A simple and efcient non-organic procedure for the isolation of genomic DNA from blood. Nucleic Acids Res. 17:8390. Loparev, V. N., M. A. Cartas, C. E. Monken, A. V. Velpandi, and A. Srinivasan. 1991. An efcient and simple method of DNA extraction from whole blood and cell lines to identify infectious agents. J. Virol. Meth. 34:105112. Malago, W. J., H. Franco, E. J. Matheucci, A. Medaglia, and F. Henrique-Silva. 2002. Large scale sex typing of ostriches using DNA extracted from feathers. BMC Biotechnol. 2:19. Miller, S., D. Dykes, and H. Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16:1215. Mozdziak, P. E., J. Angerman-Stewart, B. Rushton, S. L. Pardue, and J. N. Petitte. 2005. Isolation of chicken primordial germ cells using uorescence-activated cell sorting. Poult. Sci. 84:594600. Nakamichi, N., D. Rhoads, and D. Roufa. 1983. The Chinese Hamster Cell Emetine Resistance Gene, analysis of cDNA and genomic sequences encoding ribosomal protein S14. J. Biol. Chem. 258:1323613242. Rudbeck, L., and J. Dissing. 1998. Rapid, simple alkaline extraction of human genomic DNA from whole blood, buccal epithelial cells, semen and forensic stains for PCR. Biotechniques 25:588592.

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BAILES ET AL. E. Delany, J. Burnside, and S. Mizuno. 2000. First report on chicken genes and chromosomes 2000. Cytogenet. Cell Genet. 90:169218. Yokota, M., K. Sindo, M. Hiyoshi, I. Tsuda, and N. Tatsumi. 1998. A convenient method of DNA extraction from blood anticoagulated with EDTA. Biochem. Mol. Biol. Int. 45:617622.

Schmid, M., I. Nanda, M. Guttenbach, C. Steinlein, M. Hoehn, M. Schartl, T. Haaf, S. Weigend, R. Fries, J.-M. Buerstedde, K. Wimmers, D. W. Burt, J. Smith, S. AHara, A. Law, D. K. Grifn, N. Bumstead, J. Kaufman, P. A. Thomson, T. Burke, M. A. M. Groenen, R. P. M. A. Crooijmans, A. Vignal, V. Fillon, M. Morisson, F. Pitel, M. Tixier-Boichard, K. Ladjali-Mohammedi, J. Hillel, A. Ma ki-Tanila, H. H. Cheng, M.