an inexpensive protocol for dna extraction from blood

8/13/2019 An Inexpensive Protocol for Dna Extraction From Blood

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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY

An Inexpensive, Simple Protocol for DNA Isolation from Bloodfor 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 reli-able methods for isolating DNA from avian blood, semen,or feather pulp. The procedures are readily applicableto high-throughput 96-well plate isolation for genotypeanalysis of chicken DNA based on restriction endonucle-ase digestion or PCR. Isolation cost is primarily the costof a deep-well assay block and a few pipet tips; currentprice is less than $0.10 per sample, providing a significantcost advantage over commercial kits. The procedure em-ploys inexpensive, nonhazardous reagents and yields in-

Key words: avian blood, DNA isolation, polymerase chain reaction, restriction fragment length polymorphism

2007 Poultry Science 86:102106

INTRODUCTION

High-throughput PCR-based genotyping requiressimple methods for DNA purification. Use of DNA isola-tion kits in 96-well format to isolate sufficient DNA for50 or more PCR amplifications can be expensive, withretail prices greater than 10-fold more costly than meth-ods employing standard reagents (e.g., QIAamp, Qia-gen, Valencia, CA; or Wizard SV96, Promega Corp.,Madison, WI). Cost per sample can make high-throughput genotyping cost prohibitive for applicationto agricultural species, especially poultry. Less expen-sive commercial kits (e.g., Extract-N-Amp, Sigma-Ald-rich, St. Louis, MO) isolate DNA sufficient for only afew PCR reactions, and are therefore not suitable forgenomic analyses for many loci in genomic scans. Mostmethods for isolation of highly purified, intact DNAfrom blood use organic extractions (hazardous chemi-cals), ethanol precipitation with high-speed centrifuga-tion (not amenable to 96-well assay blocks), and numer-ous individual steps (labor intensive). An inexpensive

simple method that uses 30-min NaOH treatments hasbeen used to extract DNA from blood (Rudbeck andDissing, 1998) or feathers (Malago et al., 2002). These

2007 Poultry Science Association Inc.Received April 4, 2006.Accepted August 2, 2006.1Present address: Department of Animal and Range Sciences, South

Dakota State University, Brookings 57007.2Corresponding author: [email protected]

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tact, double-stranded DNA from as little as 2 to 10 L ofavian blood, suitable for RFLP analysis or hundreds ofPCR amplifications. We compared our method to pub-lished procedures for alkaline extraction from featherpulp and found our method to be more reliable with theadvantage of isolating intact DNA sequences that can beeasily quantified. With minor modifications, the methodcan isolate DNA for PCR genotyping from mammalianwhole blood.

techniques isolate sufficient DNA for 50 to 100 PCR reac-tions. However, the alkaline treatment also denaturesthe DNA, leaving it unsuitable for quantification by sen-sitive intercalating-dye-fluorescence or analysis by re-striction endonuclease digestion. More laborious meth-ods that isolate double-stranded DNA introduce chemi-cals (EDTA, SDS, NaI) that must be removed throughorganic extraction or ethanol precipitation before enzy-matic treatment (e.g., restriction endonuclease digestionor PCR) (Miller et al., 1988; Grimberg et al., 1989; Loparevet al., 1991; Yokota et al., 1998). An inexpensive methodhas been described whereby nuclei are purified followed

by proteinase K digestion (Ding, 1992). We have adaptedthis procedure to a 96-well format and replaced the pro-teinase K treatment to develop a protocol for isolationof double-stranded DNA sufficient for hundreds of ge-notype determinations. The cost is as little as $5 per 96-well plate. The procedure produces DNA suitable forsensitive quantification by Hoechst 33258 dye fluores-cence, PCR analysis, or restriction digestion for Southern

blot or RFLP analysis. We have applied this procedure tohigh-throughput PCR screening of avian blood samples,and for isolation from feather pulp. We have also demon-strated that our procedure can be easily modified foruse on mammalian whole blood.

MATERIALS AND METHODS

DNA Isolation

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


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tubes containing EDTA and stored frozen in microfugetubes or 96-well assay blocks, at 20C. Semen sampleswere collected by manual manipulation of males, trans-ferred to 96-well assay blocks, and frozen at 20C. TheDNA was isolated in conical 0.5-mL polypropylene 96-well assay blocks (Corning, Inc., Corning, NY). Wellswere preloaded with 0.2 to 0.5 mL of cold STM buffer(64 mMsucrose, 20 mMTris Cl, pH 7.5, 10 mMMgCl2,

and 0.5% Triton X-100) and held on ice. For high-throughput screening of frozen chicken blood, 2 to 3 Lwas added to each well with a sterile toothpick. Thesterile flat toothpick was placed about 3 to 5 mm intothe partially frozen blood and then transferred to a wellof the assay block containing STM. The toothpicks werediscarded once all wells were loaded. Larger volumesof blood (10 to 30 L) were triturated into 0.5 mL ofSTM buffer. After addition of blood, nuclei were pelletedat 1,000 g for 5 min in a centrifuge equipped to spinmicroplates. The assay block was inverted to decant thesupernatant and allowed to drain briefly on a paper

towel. Nuclear pellets were resuspended by triturationwith a multichannel pipettor to disperse the pellet into200 L of Tris-EDTA-NaCl + pronase solution(TEN+pronase; 10 mMTris-Cl, pH 8.0, 1 mMEDTA, 10mM NaCl, 100 g/mL of pronase; Sigma-Aldrich, St.Louis, MO). Precut sealing tape (Corning Inc. Life Sci-ences, Acton, MA) was used to seal the assay blocks,which were then incubated for at least 1 h (when bloodvolumes exceeded 10 L, we extended the pronase treat-ment to 2 h to ensure adequate digestion) with shakingat 37C in a bacteriological incubator, then at 65C in awater 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 ofTEN+pronase, digested for 1 h at 37C, and then inacti-vated for 10 min at 65C. Feather pulp samples weretreated in the same way as semen samples: 2 piecescomprising approximately 4 to 5 mm from the tips oftail or wing feathers were incubated for 1 h in 0.2 mLof TEN+pronase and then inactivated for 10 min at 65C.For isolation from fresh bovine blood, 100 L of bloodwas lysed in 800 L of STM. The first nuclear pellet wassuspended in a new aliquot of STM buffer, and thennuclei were re-pelleted. The nuclear pellet was then sus-pended 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; Ma-lago et al., 2002). Feather tips (approximately 5 mm as2 pieces) were incubated in 50 L of 100 mMNaOH for1 h at 37C and then neutralized with 240 L of 40 mMTrisCl, pH 7.5.

Isolated DNA samples were stored frozen at 20C.Final DNA quantification was by Hoechst 33258 fluo-

rescence measured in a fluorometer (model TKO, HoeferScientific Instruments, San Francisco, CA) according tomanufacturers protocols.

Table 1. Yield of DNA from a variety of sources1

Yield (g)

Volume AverageSource (L) n Range SD

Chicken blood, frozen Toothpick 25 1.6 to 88 20 22Chicken blood, fresh 2 2 25 to 30

5 2 40 to 4110 2 40 to 6020 2 100 to 112

30 2 125 to 147Chicken semen, frozen 5 9 15 to 40 26 8

10 6 30 to 40 34 4Chicken feather tip 20 2 to 10Bovine blood, frozen 200 3 3 to 5

1The indicated volumes of sample from the indicated source wereprocessed according to our described procedures (Materials and Meth-ods). Foreach sourceand volume, thenumber of samples(n) is indicatedas well as the range of DNA yield. Where 3 or more samples wereprocessed, the average yield and standard deviation (SD) were calcu-lated.

Restriction Endonuclease Digestion

Genomic DNA samples (3 to 4 g) were digested in50-L reactions using buffers and conditions recom-mended by the enzyme supplier (Promega Corp., Madi-son, WI). After digestion, DNA samples were resolvedin 0.7 or 1.5% agarose gels in 0.5Tris borate EDTA (50mMTris borate, pH 8.3, 1 mMNa2EDTA), stained withethidium bromide, and detected by fluorescence (532nm excitation, 610 nm emission) with a Typhoon 9600scanner (GE Health Care, Piscataway, NY). Selected gelswere further analyzed by Southern blot hybridization(Nakamichi et al., 1983).

PCR Genotyping

Microsatellite genotype determination used fluores-cent-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 (ERgene on chicken chromosome 3 (unpublished); KS001(5-FAM-gatcattgctgcaaaatgga-3 , 5-gaaggtgactcagat-tagg-3) is specific for a locus on chicken chromosome17 (unpublished); and LEI0217 (5- HEX-gatgactgaga-gaaataacttg-3 , 5-aaattactgaggcacaggag-3 ) andUMA1.070 (5- HEX-aagcttttaaaccaatctga-3, 5-tcctgcatg-tgccctttgta-3) derive from chicken chromosome 1(Schmid et al., 2000). The DNA samples (1 to 2 L) wereamplified in 20-L PCR reactions in 200-L 96-well PCRplates (VWR International, Bristol, CT) in a thermocycler(model PTC-100, MJ Research, Watertown, MA). EachPCR reaction contained 1 PCR buffer (50 mM TrisCl,pH 8.3, 1 mMMgCl2, 30 mg/mL of BSA), 0.2 mMdNTP,0.2 M of microsatellite primers, and 5 U ofTaq polymer-ase. Reactions were either overlaid with 20 to 50 L ofmineral oil, or sealed with tape. The PCR conditionswere 90C for 2 min, 45 cycles of 90C for 30 s, 45C for1 min, 72C for 1 min, followed by 72C extension for 7min. Products were either resolved on 30 40 cm 6%


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Figure1. Comparison of DNA restrictability: Southern blot ofHindIIIdigested DNA samples (2 g/lane) probed with a cDNA for estrogenreceptor . Lanes 1 to 3: DNA samples prepared by our method; lanes4 to 5: DNA samples purified by standard methods. Molecular weights

based on DNA standards are indicated to the right of the figure.

acrylamide denaturing gels, then detected with a Ty-phoon 9600 scanner, or resolved and detected on anABI377 sequencer (model ABI 377, Applied Biosystems,Foster City, CA). For the ABI377 sequencer, PCR prod-ucts were diluted 1:5 with water then 1 L of the dilutionwas mixed with 3 L of TAMRA-labeled internal linestandard (Applied Biosystems) denatured at 100C for2 min, chilled on ice, and samples (2 L) were loadedand resolved as recommended by the manufacturer. Al-lele sizes were determined using Genescan software(Applied Biosystems).

The PCR analysis for Wxho and Ribo sex determina-tion 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 aprevious method based on cell lysis in hypotonic, non-ionic detergent, low-speed centrifugation to pellet nu-clei, disruption of nuclei, and partial proteinase K diges-tion in a low ionic strength buffer (Ding, 1992). We modi-fied this technique for use in 96-well format and usedpronase E, a protease that can be denatured at 65C,thus maintaining the native, double-strand form of theDNA. We evaluated the DNA yield from fresh or frozenchicken blood, chicken semen, chicken feather pulp, and

bovine blood (Table 1). As can be seen from the data inTable 1, there is a nearly linear relationship betweenisolated DNA concentration and chicken blood volumeusing our procedure. The quantity of DNA is sufficientfor hundreds of standard PCR genotype analyses (e.g.,microsatellite and single nucleotide polymorphismanalysis).

Our procedure yields intact DNA sufficiently pure forrestriction endonuclease treatment. We compared DNAisolated from chicken blood by our method to DNApurified using standard SDS lysis, proteinase K diges-

Figure 2.Polymerase chain reaction sexing of DNA samples isolated

by alkaline extraction or by pronase digestion. The DNA samples weresubjected to PCR using the WXho and Ribo primers, and resolved ina 1.5% agarose gel. Top: 24 DNAsamples extracted from feather pulp bythe NaOH method from different hens. Bottom: DNAsamples extractedfrom feather pulp by TEN+pronase (Tris-EDTA-NaCl + pronase) diges-tion from the same hens as in the top panel.

tion, organic extraction, and ethanol precipitation(Grimberg et al., 1989). The DNA aliquots were digestedwith various enzymes (AluI, BamHI, EcoRI, or HindIII).Electrophoretic patterns from digested DNA were indis-tinguishable between the DNA isolated by the 2 differentmethods (data not shown). Southern blot analysis using

a cDNA probe to ER (provided by D. Bunik, Universityof Illinois, Urbana-Champaign) showed no differences

between the hybridization patterns (Figure 1). Thus, thismethod inactivates nonspecific nucleases, introduces noinhibitory compounds, and produces DNA solutionssuitable for restriction endonuclease digestion for South-ern blot analysis and RFLP genotyping.

To evaluate the DNA isolation procedure for high-throughput PCR genotyping, we isolated DNA from 886frozenblood samples archived for over 1 yr in a frost-freefreezer using our toothpick method. Typically, bloodsamples 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 sam-ples showed a variable range of DNA concentrations(Table 1). Given these concentrations, we used 2 L ofthe DNA solution in 20-L PCR amplifications for geno-typing at 2 microsatellite loci, ADR001 and KS001. Forthe 886 archived chicken blood samples, we obtainedgenotype data for 792 samples for ADR001 and 737 sam-ples for KS001, where 78 samples amplified for ADR001only, 28 samples amplified for KS001 only, and 68 sam-ples gave no amplification for either locus. The latterare most likely because of low DNA quantities for thosesamples or pipetting errors. Therefore, using our rapid


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RESEARCH NOTE 105

Figure 3.Microsatellite PCR analysis of DNA samples isolated by alkaline extraction or by pronase digestion. The DNA samples were amplifiedfor ADR001 (upper panel) and KS001 (lower panel). Lanes 1 to 10: DNA extracted from feather pulp by NaOH treatment; lane 11: DNA purifiedfrom blood by standard methods; lanes 12 to 21: DNA extracted from feather pulp by TEN+pronase (Tris-EDTA-NaCl + pronase) digestion; lanes22 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 818out 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 de-terminations (data not shown). We compared DNA iso-lated by our method from single feathers to DNA ex-tracted using alkaline treatment (Rudbeck and Dissing,1998; Malago et al., 2002). Figure 2 shows PCR datacomparing these DNA samples when amplified withWXho and Ribo sexing primers. Only 9 of the 24 DNAsamples isolated by NaOH extraction yielded adequateamplification, whereas all 24 of the samples isolated byTEN+pronase digestion amplified, with 1 DNA sampleproducing only weak amplification, and 2 amplifying

only the W-specific fragment. We also tested these DNAsamples for microsatellite genotyping with ADR001 andKS001 and found that 9 of 10 TEN+pronase DNA sam-ples amplified for both loci whereas zero of 10 NaOH-extracted DNA samples amplified for either locus (Fig-ure 3). We tested these same DNA samples for 2 addi-tional microsatellite loci (LEI0217 and UMA1.070) andproduced amplifications for 3 of 10 NaOH-extractedDNA samples vs. 10 of 10 TEN+pronase-extracted DNAsamples (data not shown). Therefore, in our experience,the TEN+pronase digestion is much more reliable andproduces DNA more amenable to PCR genotyping.

We adapted the procedure to work with mammalian

blood samples. Based on the low frequency of nucleatedcells in mammalian whole blood and greater startingsample volumes, we found that 2 successive STM treat-ments were required to reduce contaminating proteinsand avoid coagulated proteins upon enzyme denatur-ation at 65C. When we used 2 successive centrifugationsteps we obtained final DNA that could be successfullyamplified in PCR using primers to bovine HSP70 (pro-vided by C. Rosenkrans, University of Arkansas, Fay-etteville; data not shown).

This simple, and inexpensive method for isolation ofDNA from a variety of sources will facilitate the rapid,

high-throughput PCR or RFLP analysis of genotypes inavian and other species in situations where overall costs

must be minimized.

ACKNOWLEDGMENTS

This work was partially supported by grants to JDKand DDR from the Arkansas Biosciences Institute, andfrom Cobb-Vantress Inc., Siloam Springs, AR. KatrinaCollins, and Laura Thorne provided valuable input andtechnical assistance. Special thanks to Bobbi Okimoto forassistance and training in the UA DNA Core Laboratory.

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Grimberg, J., S. Nawoschik, L. Belluscio, R. McKee, A. Turck,and A. Eisenberg. 1989. A simple and efficient non-organicprocedure 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 efficient and simple method ofDNA extraction from whole blood and cell lines to identifyinfectious 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 ostrichesusing DNA extracted from feathers. BMC Biotechnol. 2:19.

Miller, S., D. Dykes, and H. Polesky. 1988. A simple saltingout procedure for extracting DNA from human nucleatedcells. 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 germcells using fluorescence-activated cell sorting. Poult. Sci.84:594600.

Nakamichi, N., D. Rhoads, and D. Roufa. 1983. The ChineseHamster Cell Emetine Resistance Gene, analysis of cDNAand genomic sequences encoding ribosomal protein S14. J.Biol. Chem. 258:1323613242.

Rudbeck, L., and J. Dissing. 1998. Rapid, simple alkaline extrac-tion of human genomic DNA from whole blood, buccalepithelial cells, semen and forensic stains for PCR. Biotech-niques 25:588592.


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an inexpensive protocol for dna extraction from blood

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