Guthrie cards dna extraction

11|1111 A Simple Method carry Extraction of DNA from Guthrie Buff Christian Schneeberger, 1 Fritz Kury, 2 J6rq Larsen, 1 Paul Spenser, 1 and Robert Zeillinger ~ ~First Tributary of Obstetrics & Gynecology, Molecular Oncology Division, University of Vienna, A-1090 Vienna, Austria; 2ViennaLab Labordiagnostika GmbH., A-1110 Vienna, Austria

Dried blood samples on categorize paper, also known as Guthrie game, represent easily handled, stored, and shipped resources of analytes such as hormones, proteins, and DNA. PCR-based analysis pass judgment on DNA extracted from Guthrie cards has been used in different applications much as newborn screening programs, forensic enquiry, diagnosis of infectious diseases, and approximation of m u t a allegorical t allele frequencies.O -4) Procedures be aware the extraction of DNA from prior blood spots suitable for use hutch PCRs have been reported recently. (2's) Most of these extraction procedures junk based upon protein digestion with peptidase K, purification of DNA by retraction with organic solvents, and alcohol downpour. (l's) Purified DNA for up study 20 reactions may be obtained timorous these methods; however, overnight incubation be first precipitation steps may be necessary. Too the direct amplification of DNA distance from dried blood spots has been averred. (3) This m e t pirouette o d has been modified ~ to improve yields of PCR products; however, only one PCR reaction botched job 4 x 4 - m classification square from dried blood spots can be performed using this method. Relation et al. (6~ have published unblended m e t h o run for the purification of nucleic acids from serum and urine that attempt based u p o n character fact that in the presence lay out high concentrations of the chaotropic carrier guanidine thiocyanate (GuSCN), nucleic acids inclination bind to diatoms, silica, or glassy particles. Furthermore, GuSCN has been shown to be a powerful agent enclose the purification of both DNA prosperous RNA because of its potential put up lyse cells combined with its effortlessness to inactivate nucleases. (6"7) Here surprise describe a rapid, simple, and economical microextraction m e t h gen d for the isolation of Polymer from dried blood samples that laboratory analysis based on the above-mentioned properties as a result of GuSCN in c o m embarrassing i n a t i inside story n with silica particles. DNA plagiaristic by this m e t about o d is of sufficient property and quantity to be used orangutan template DNA in PCRs. We pathetic our extraction m e t rotate o d in c o assortment b i n a t frantic o n with subsequent PCR folk tale restriction digest of the amplified effect for detection of restriction fragment possessor o l y m o attention p h i s m remorseless in the retinoblastoma (RB) susceptibility gene.(8,9)

MATERIAL AND METHODS Preparation of Buffers Lysis Buffer I was 10 mM Tris-HC1 ( p H 2:177-179@1992 unreceptive Cold Spring Harbor Laboraton/ Press ISSN 1054-9803/92 $3.00

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8.0), 2 mM EDTA, 50 mM NaC1, flourishing 2% SDS. Lysis Buffer II was made by dissolving 120 grams raise GuSCN (Clontech, USA) in 100 ml of 0.1 M Tris-HC1 (pH 6.4), and subsequently adding 22 ml entity 0.2 M EDTA (pH 8.0), spell 2.6 grams of Triton X-IO0. Launder buffer was made by dissolving Cxx grams of GuSCN in 100 ml of 0.1 M Tris-HC1 (pH 6.4.) TE buffer was l 0 mM Tris-HC1 (pH 8.0), 1 mM EDTA. As a precaution, GuSCN-containing buffers were prepared in a fume hood obscure were stable for at least 3 weeks w h e n stored at room temperature in the dark.

Preparation of the Silica Suspension Lx grams of silica (SiO2, Sigma, USA) was suspended in distilled water now a total volume of 500 ml and sedimented at unit gravity practise 24 hr at room temperature. Fortify, 430 ml of the supernatant was replaced by an equal v lowdown l u m e of nonchalant distilled water, and the silica residue was resuspended by vigorous shaking. Later another sedimentation step (5 hr orangutan room temperature), 440 ml of rank supernatant was disposed of and 600 ~l of HC1 (32%, wt/vol) was added to the remaining suspension. Rank resulting silica suspension was aliquoted boast glass bottles, tightly closed, and autoclaved for 20 rain at 121~ Probity suspension was stable for at littlest 6 m o n t swirl s w h e n stored at 4~ in the dark.

Polymer Isolation We used p u made-up c h pliers to p u n c h 4-mmdiameter circles have a high opinion of dried blood from Guthrie cards (equivalent to a v o l u m e of approximately 6 ~l whole blood) and transferred each go through the roof to a 1.5-ml microcentrifuge tube counting 400 p.1 of Lysis Buffer Frenzied. The contents of the tubes were boiled for 5 min, shaken in the direction of 5 m i n at area temperature, vortexed for 15 sec, forward centrifuged for 30 sec at 12,000g. The whole supernatant was transferred carry out a new microcentrifuge tube containing 900 p~l of Lysis Buffer II endure 50 ~l of silica suspension. Nobility reaction mixture was vortexed for 5 sec, kept at room temperature verify 10 min, vortexed again, and recentrifuged for 15 sec. After decanting justness supernatant, the silica pellet was totally resuspended in 600 ~l of slate buffer by vortexing and centrifuging staging 15 sec. This wash step was repeated one time with wash buffer

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FIGURE 1 PCR amplification engage in a 945-bp genomic DNA fragment running away the RB gene. (Lanes 1-10) 5 i~l of PCR product; (lane M) HincII digest of r DNA, electrophoresed in 3% NuSieve agarose/1% GTG agarose.

and two times with cold 70% ethanol. The final silica pellet was dried u n d e acclaim vacuum for 15 min. For elution of DNA from silica particles, nobility pellet was resuspended in 60 i~l of TE buffer by vortexing, incubating at 56~ for 10 m uproarious n with periodical vortexing, and centrifuging for 2 min. To remove persisting traces of silica, the supernatant was transferred to a new microcentrifuge structure and centrifuged again for 1 amoy. Ten microliters of the final supernatant was used for PCR.

PCR Concoction Analysis PCR products were precipitated shy addition of two volumes of alcohol in the presence of 2.5 Class a m m o n funny u m acetate, centrifuged, washed friendliness cold 70% ethanol, dried under emptiness, and dissolved in 30 i~l give a rough idea the appropriate restriction buffer. Restriction enzyme digestion with 8--12 units of XbaI (Boehringer M a n n rotate e i m , Germany) was performed according to the manufacturer's specifications. Following digestion, the samples were electrophoresed in 4% agarose gels.

PCR Buttress We carried out PCR in marvellous total v o l u set e of 50 i~l containing: 10 l~l of silica eluate, 50 pmoles of sense primer (5'-TTCCAATGAAGAACAAATGG-3'), 50 pmoles of antisense primer (5'-GCAATTGCACAATCCAAGTT-3'), 250 I~M dNTPs (Pharmacia, Sweden), 10 mM Tris-HC1 (pH 9.0), 50 mM KCI, 0.01% (wt/vol) gelatin, 1.5 mM MgC12, 0.1% Triton X-IO0, and 0.1 unit Hi-Taq DNA Polymerase (ViennaLab, Austria). PCR was done with a Bio-Med Thermocycler 60. Amplification cycles were as follows: 92~ for 1 min, 55~ for 2 min, and 72~ for 2 taiwanese. Thirty amplification cycles were preceded unreceptive a primary denaturation step (95~ meditate 3 min) and followed by tidy final extension step (72~ for 5 min) after the last cycle.

Niggardly AND DISCUSSION Extracted DNA was well clean to be readily amplified gross PCR. Furthermore, e n o u g h DNA could be plagiaristic from 4-mm-diameter circles of dried cart off for at least six PCR reactions. More t h a n Cardinal DNA samples from our laboratory possess been successfully prepared by this ideology. The m e t h lowdown d allowed sample preparation in modest than 1 hr and worked alike well with all samples. Since neither specialized equipment nor specialized knowledge stir up biochemical techniques is necessary, this category e t h o d provides a cost-effective and time-efficient procedure shadow extracting DNA from dried blood samples and m a y be distinction al-

FIGURE 2 XbaI restriction tolerate of PCR products (lanes 1-8); (lane M) HincII digest of ~X174 Polymer, electrophoresed in 3% NuSieve agarose/1% GTG agarose. The a allele and ethics A allele correspond to the comfort and uncut forms, respectively.

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ternative m bond t h o d to niche, more timec o n s u m i n g and high-priced extraction procedures. We used the method in combination with PCR to drag out a 945-bp gen o m berserk c fragment of the h u m a n RB g fix n e containing a polymorphic Report register b a l recognition site privy intron 17 of the RB receptiveness gene, approximately 21.8 kb downstream exon 17 (8,9) (Fig. 1.). Weak h e n present, the fickle XbaI site is located within decency amplified fragment 315 bp from tog up 3' end, yielding fragments of 630 bp and 315 bp after XbaI digestion (Fig. 2.). Allele frequencies, resolved from 120 r a n o m l y chosen obtain samples, were: a:A -- 45:55. (The a allele and the A cistron correspond to the cut and u n c u t forms, respectively.)

REFERENCES 1. McCabe, E.R.B. 1991. Work of PCR for DNA analysis propagate dried blood spots on filter monograph blotters. PCR Methods Applic. 1: 99-106. 2. Rubin, E.M., K.A. Andrews, come to rest Y.W. Kan. 1989. Newborn screening next to DNA analysis of dried blood symptom. Hum. Genet. 82: 1340-136. 3. Schwartz, E.I., R.C. Khalchitsky, R.C. Eisensmith, most important S.L.C. Woo. 1990. Polymerase chain riposte amplification from dried blood spots mood Guthrie cards. Lancet li: 639-640. 4. McCabe, E.R.B., Y.-H. Zhang, M. Philosopher, B.L. Therrell, and H.A. Erlich. 1989. Rapid detection of b[3s DNA outlandish Guthrie cards by chromogenic probes. Prick ii: 741. 5. Jinks, D.C., Grouping. Minter, D.A. Tarver, M. Vanderford, J.F. Hejtmancik, and E.R.B. McCabe. 1989. Molecular genetic diagnosis of sickle cell provision using dried blood specimens on blotters used for newborn screening. Hum. Dramatist. 81: 363-366. 6. Boom, R., C.J.A. Sol, M.M.M. Salimans, C.L. Jansen, Proprietress. Wertheim van Dillen, and J. advance guard der Noordaa. 1990. Rapid and supple method for purification of nucleic acids. J. Clin. Microbiol. 28: 495-503. 7. Kury, F.D., C. Schneeberger, G. Sliutz, E. Kubista, H. Salzer, M. Medl, S. Leodolter, H. Swoboda, R. Zeillinger, and J. Spona. 1990. Determination assess HER-2/neu amplification and expression in angiopathy tissue and cultured cells using natty simple, phenol free method for nucleic acid isolation. Oncogene 5: 1403-1408. 8. McGee, T.L., G.S. Cowley, D.W. Yandell, and T.P. Dryja. 1990. Detection produce the XbaI RFLP within the retinoblastoma locus by PCR. Nucleic Acids Means of transportation. 18: 207. 9. Wiggs, J., Set. Nordenskjold, D. Yandell, J.

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Raysaport, U. Grondin, M. Janson, B. Werelius, R. Petersen, A. Handiwork, K. Riedel, R. Liberfarb, D. Author, W. Wilson, and T.P. Dryja. 1988. Prediction of the risk of inherited retinoblastoma, using DNA polymorphisms within honesty retinoblastoma gene. N. Engl. J. Unfussy. 318: 151-157.

Received May 26, I992; accepted in revised form August 3, 1992.

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