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Poult Sci 2007. 86:102-106
© 2007 Poultry Science Association
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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY: Research Note

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{dagger}, J. D. Kirby{dagger},{ddagger},1 and D. D. Rhoads{dagger},§,2

* Missouri State Highway Patrol Crime Laboratory, Jefferson City 65101; {dagger} Program in Cell and Molecular Biology; {ddagger} Department of Poultry Science; and § Department of Biological Sciences, University of Arkansas, Fayetteville 72701

2 Corresponding author: drhoads{at}uark.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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 significant cost advantage over commercial kits. The procedure employs inexpensive, nonhazardous reagents and yields intact, double-stranded DNA from as little as 2 to 10 µL of avian blood, suitable for RFLP analysis or hundreds of PCR amplifications. 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 quantified. With minor modifications, 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
High-throughput PCR-based genotyping requires simple methods for DNA purification. Use of DNA isolation kits in 96-well format to isolate sufficient DNA for 50 or more PCR amplifications 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 high-throughput 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 sufficient 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 purified, 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 sufficient DNA for 50 to 100 PCR reactions. However, the alkaline treatment also denatures the DNA, leaving it unsuitable for quantification by sensitive intercalating-dye-fluorescence 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 purified 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 sufficient for hundreds of genotype determinations. The cost is as little as $5 per 96-well plate. The procedure produces DNA suitable for sensitive quantification by Hoechst 33258 dye fluorescence, 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 modified for use on mammalian whole blood.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
DNA Isolation

Whole blood samples were collected via Vacutainer (Becton Dickinson, Franklin Lakes, NJ) blood collection tubes containing EDTA and stored frozen in microfuge tubes or 96-well assay blocks, at –20°C. Semen samples were collected by manual manipulation of males, transferred to 96-well assay blocks, and frozen at –20°C. The DNA was isolated in conical 0.5-mL polypropylene 96-well 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 high-throughput screening of frozen chicken blood, 2 to 3 µL was added to each well with a sterile toothpick. The sterile flat 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 µL) were triturated into 0.5 mL of STM buffer. After addition of blood, nuclei were pelleted at 1,000 x g for 5 min in a centrifuge equipped to spin microplates. The assay block was inverted to decant the supernatant and allowed to drain briefly on a paper towel. Nuclear pellets were resuspended by trituration with a multichannel pipettor to disperse the pellet into 200 µL of Tris-EDTA-NaCl + pronase solution (TEN+pronase; 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 10 mM NaCl, 100 µg/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 µL, we extended the pronase treatment to 2 h to ensure adequate digestion) with shaking at 37°C in a bacteriological incubator, then at 65°C 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 µL) was triturated into 0.2 mL of TEN+pronase, digested for 1 h at 37°C, and then inactivated for 10 min at 65°C. 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 65°C. For isolation from fresh bovine blood, 100 µL of blood was lysed in 800 µL of STM. The first 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 µL of TEN+pronase, digested for 1 h at 37°C, and then inactivated at 65°C 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 µL of 100 mM NaOH for 1 h at 37°C and then neutralized with 240 µL of 40 mM TrisCl, pH 7.5.

Isolated DNA samples were stored frozen at –20°C.

Final DNA quantification was by Hoechst 33258 fluorescence measured in a fluorometer (model TKO, Hoefer Scientific Instruments, San Francisco, CA) according to manufacturer’s protocols.

Restriction Endonuclease Digestion

Genomic DNA samples (3 to 4 µg) were digested in 50-µL 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 0.5x Tris borate EDTA (50 mM Tris borate, pH 8.3, 1 mM Na2EDTA), stained with ethidium bromide, and detected by fluorescence (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 fluorescent-labeled primers (custom synthesis, MWG Biotech, High Point, NC). We developed primer ADR001 (5'-HEX-gcttcgactatctagaatg-3', 5'-gctaaaatataaaatgcagg-3') as a specific marker for the estrogen receptor {alpha} (ER{alpha} gene on chicken chromosome 3 (unpublished); KS001 (5'-FAM-gatcattgctgcaaaatgga-3', 5'-gaaggtgactcagat-tagg-3') is specific for a locus on chicken chromosome 17 (unpublished); and LEI0217 (5'- HEX-gatgactgaga-gaaataacttg-3', 5'-aaattactgaggcacaggag-3') and UMA1.070 (5'- HEX-aagcttttaaaccaatctga-3', 5'-tcctgcatg-tgccctttgta-3') derive from chicken chromosome 1 (Schmid et al., 2000). The DNA samples (1 to 2 µL) were amplified in 20-µL PCR reactions in 200-µL 96-well PCR plates (VWR International, Bristol, CT) in a thermocycler (model PTC-100, MJ Research, Watertown, MA). Each PCR reaction contained 1x PCR buffer (50 mM TrisCl, pH 8.3, 1 mM MgCl2, 30 mg/mL of BSA), 0.2 mM dNTP, 0.2 µM of microsatellite primers, and 5 U of Taq polymerase. Reactions were either overlaid with 20 to 50 µL of mineral oil, or sealed with tape. The PCR conditions were 90°C for 2 min, 45 cycles of 90°C for 30 s, 45°C for 1 min, 72°C for 1 min, followed by 72°C extension for 7 min. Products were either resolved on 30 x 40 cm 6% 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 µL of the dilution was mixed with 3 µL of TAMRA-labeled internal line standard (Applied Biosystems) denatured at 100°C for 2 min, chilled on ice, and samples (2 µL) 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Our DNA isolation technique was developed from a previous method based on cell lysis in hypotonic, non-ionic detergent, low-speed centrifugation to pellet nuclei, disruption of nuclei, and partial proteinase K digestion in a low ionic strength buffer (Ding, 1992). We modified this technique for use in 96-well format and used pronase E, a protease that can be denatured at 65°C, 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 1Go). As can be seen from the data in Table 1Go, there is a nearly linear relationship between isolated DNA concentration and chicken blood volume using our procedure. The quantity of DNA is sufficient for hundreds of standard PCR genotype analyses (e.g., microsatellite and single nucleotide polymorphism analysis).


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  		Table 1. Yield of DNA from a variety of sources1
 
Our procedure yields intact DNA sufficiently pure for restriction endonuclease treatment. We compared DNA isolated from chicken blood by our method to DNA purified using standard SDS lysis, proteinase K digestion, 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{alpha} (provided by D. Bunik, University of Illinois, Urbana-Champaign) showed no differences between the hybridization patterns (Figure 1Go). Thus, this method inactivates nonspecific nucleases, introduces no inhibitory compounds, and produces DNA solutions suitable for restriction endonuclease digestion for Southern blot analysis and RFLP genotyping.


Figure 1
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  		Figure 1. Comparison of DNA restrictability: Southern blot of HindIII digested DNA samples (2 µg/lane) probed with a cDNA for estrogen receptor{alpha}. Lanes 1 to 3: DNA samples prepared by our method; lanes 4 to 5: DNA samples purified by standard methods. Molecular weights based on DNA standards are indicated to the right of the figure.

 
To evaluate the DNA isolation procedure for high-throughput 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 difficult to pipet when thawed (clots). After DNA isolation, quantification of 25 random samples showed a variable range of DNA concentrations (Table 1Go). Given these concentrations, we used 2 µL of the DNA solution in 20-µL PCR amplifications 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 amplified for ADR001 only, 28 samples amplified for KS001 only, and 68 samples gave no amplification for either locus. The latter are most likely because of low DNA quantities for those samples or pipetting errors. Therefore, using our rapid 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 2Go shows PCR data comparing these DNA samples when amplified with WXho and Ribo sexing primers. Only 9 of the 24 DNA samples isolated by NaOH extraction yielded adequate amplification, whereas all 24 of the samples isolated by TEN+pronase digestion amplified, with 1 DNA sample producing only weak amplification, and 2 amplifying only the W-specific fragment. We also tested these DNA samples for microsatellite genotyping with ADR001 and KS001 and found that 9 of 10 TEN+pronase DNA samples amplified for both loci whereas zero of 10 NaOH-extracted DNA samples amplified for either locus (Figure 3Go). We tested these same DNA samples for 2 additional microsatellite loci (LEI0217 and UMA1.070) and produced amplifications 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.


Figure 2
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  		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.

 

Figure 3
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  		Figure 3. Microsatellite PCR analysis of DNA samples isolated by alkaline extraction or by pronase digestion. The DNA samples were amplified for ADR001 (upper panel) and KS001 (lower panel). Lanes 1 to 10: DNA extracted from feather pulp by NaOH treatment; lane 11: DNA purified 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.

 
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 65°C. When we used 2 successive centrifugation steps we obtained final DNA that could be successfully amplified 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.


   FOOTNOTES
 
1 Present address: Department of Animal and Range Sciences, South Dakota State University, Brookings 57007. Back

Received for publication April 4, 2006. Accepted for publication August 2, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Ding, Y. 1992. DNA preparation from trace forensic biological evidences. Pages 371–375 in Handbook of PCR Gene Amplification 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 efficient non-organic procedure for the isolation of genomic DNA from blood. Nucleic Acids Res. 17:8390.[Free Full Text]

Loparev, V. N., M. A. Cartas, C. E. Monken, A. V. Velpandi, and A. Srinivasan. 1991. An efficient and simple method of DNA extraction from whole blood and cell lines to identify infectious agents. J. Virol. Meth. 34:105–112.[Web of Science][Medline]

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.[Medline]

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.[Free Full Text]

Mozdziak, P. E., J. Angerman-Stewart, B. Rushton, S. L. Pardue, and J. N. Petitte. 2005. Isolation of chicken primordial germ cells using fluorescence-activated cell sorting. Poult. Sci. 84:594–600.[Abstract/Free Full Text]

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. 258:13236–13242.[Abstract/Free Full Text]

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 25:588–592.[Web of Science][Medline]

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. A’Hara, A. Law, D. K. Griffin, 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. Mäki-Tanila, H. H. Cheng, M. E. Delany, J. Burnside, and S. Mizuno. 2000. First report on chicken genes and chromosomes 2000. Cytogenet. Cell Genet. 90:169–218.

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. 45:617–622.[Web of Science][Medline]




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