Ballvora, A.

Gebhardt, C.

Klemm, M.

Matzner, U.

Rickert, A.

English

MPG.PuRe

Biotechnol. Appl. Biochem. (2005) 42, 93–96 (Printed in Great Britain) doi:10.1042/BA20040177 93Quantitative genotyping of single-nucleotide polymorphismsby allele-specific oligonucleotide hybridization on DNAmicroarraysAndreas M. Rickert*, Agim Ballvora*1, Ulrich Matzner†, Manfred Klemm†and Christiane Gebhardt**Max-Planck-Institute for Breeding Research, Carl-von-Linne-Weg 10, 50829 Cologne, Germany, and†Alpha Technology GmbH, Hu¨rth, GermanyGenotyping of SNPs (single-nucleotide polymorph-isms) has challenged the development of several noveltechniques [Twyman and Primrose (2003) Pharmaco-genomics 4, 67–79]. Most of these methods have beenintroduced to discriminate binary SNPs in diploidspecies. In the present study, the quantitative geno-typing of SNPs in natural DNA pools of a polyploidorganism via DNA microarrays was analysed. Threerandomly selected SNP loci were genotyped in thetetraploid species potato (Solanum tuberosum). Foreach SNP, 24 oligomers were designed, 12 with forwardand 12 with reverse orientation. They contained thepolymorphic site at one of the positions 11, 14 and17. Several steps of optimizations were performed,including the ‘materials’ used and the establishmentof hybridization conditions. Glass surfaces were eitherepoxy- or aldehyde-modified, and allele-specific oligo-nucleotides contained either SH or NH2 groups. Hy-bridization stringency conditions were established byvarying the concentration of formamide in the hybrid-ization buffer. For SNP BA213c14t7/403, the quanti-tative discrimination between all four different natur-ally occurring genotypes could be demonstrated.IntroductionSNPs (single-nucleotide polymorphisms) represent the mostcommon type of genetic variations accounting for approx.90% of all polymorphisms [2]. As a result of genome se-quencing efforts, large number of SNPs have been dis-covered, not only in humans [3] (see also the SNP Consor-tium LTP at http//snp.cshl.org), but also in other diploidorganisms such as Drosophila melanogaster (fruitfly or vinegarfly) [5], Arabidopsis thaliana (thale cress) [6] and cerealspecies [7], and recently in the polyploid potato Solanumtuberosum [8]. Owing to their direct accessibility, SNPshave become popular markers for a wide range of geneticapplications, and a number of different SNP genotypingmethods have been introduced. However, no single methodhas been widely accepted [1,9–14]. For linkage and associ-ation studies, many SNP markers have to be scored inlarge number of individuals. Since this procedure is time-consuming and costly, an alternative approach is to poolsamples of affected and unaffected individuals and sub-sequently estimate, quantitatively, allele frequencies in thesesubsets. To attain this goal, several SNP genotyping methodshave been evaluated [15–28]. Although ASO (allele-specificoligonucleotide) hybridization on microarrays is amongthe most promising techniques for genotyping individualSNPs [6,29], documented studies of its applicability forquantification of allele frequencies in DNA pools are lacking.In the present paper we describe quantitative genotypingusing microarray-based ASO hybridization of three binarySNPs in different homozygous and heterozygous tetraploidpotatoes. These genotypes represent natural pools of fouralleles for which five allelic ratios are distinguishable: 4:0, 3:1,2:2, 1:3 and 0:4 [25].Materials and methodsPlant materialF1 plants of a cross between two tetraploid potato cul-tivars SR11 and SR12 were provided by SAKA-RAGIS(Pflanzenzucht GbR, Windeby, Germany) [8]. F1 plants thathad been evaluated by pyrosequencing or SNuPE (singlenucleotide primer extension) combined with DHPLC (de-naturing high-performance liquid chromatography) for thetest of SNPs and represented the different allele dosages,were selected for the evaluation of quantitative accuracy ofallele-specific hybridization on microarrays. Genomic DNAwas isolated from leaf tissue as described previously [30].SNP markersThe SNPs BA44a10t7/174, BA213c14t3/268 + 269 andBA213c14t7/403 have been identified by comparative SangerKey words: allele-specific-oligonucleotide, DNA-microarray, locus, polyploidy,quantitative genotyping, single-nucleotide polymorphism.Abbreviations used: ASO, allele-specific oligonucleotide; DHPLC, denaturinghigh-performance liquid chromatography; SNP, single-nucleotidepolymorphism; SNuPE, single nucleotide primer extension.1 To whom correspondence should be addressed (emailballvora@mpiz-koeln.mpg.de).C© 2005 Portland Press Ltd94 A. M. Rickert and othersFigure 1 Schematic representation of the panel of ASOs designed forgenotyping the A ↔ T-SNP BA213c14t7/403The set of ASOs for allele-specific hybridization comprises 24 different22-meric oligonucleotides querying the sense as well as the antisense strandof the PCR-amplified target region including the SNP. ASO probes are walkingover the polymorphic sites presenting the allelic nucleotide at position 11, 14or 17 from the 5′-end. For each of these ASOs, four different versions weredesigned, carrying all four bases at the SNP position. ASOs are attached totails composed of a C6 linker and an SH or NH2 group. N, A/T/G/C; W, A/T.DNA sequencing and can be accessed through the websitehttp://gabi.rzpd.de/PoMaMo.html [8]. The precise alleledosages of SNPs in the different genotypes were confirmedby pyrosequencing [25] or by SNuPE in combination withDHPLC as described previously [31].Amplification and labelling of target sequencesFor amplification and labelling of target sequences (lengthsbetween 180 and 216 bp), the following primer combi-nations were used: BA44a10t7Fa1 (5′-CTACATCACCTGC-AGCACTCC-3′) and BA44a10t7Ra1 (5′-TTTCATGGAA-GCTGGACGGCTAG-3′); BA213c14t3Ca1 (5′-CGGTCA-GGGACATCATGCGCA-3′) and BA213c14t3Da1 (5′-GT-GGTATCTGCTAGCAATTGCTAC-3′); BA213c14t7Ba1rev(5′-TCTAACCTGGGCAGTCAGAGAC-3′) and BA213-c14t7B (5′-TCTTGACGCAAACCTCTGCGAG-3′). Primerswere purchased from Metabion (Martinsried, Germany).PCRs were performed as described in [25]. Aliquots (30 ng)of the initial PCR amplifications were subjected to a secondround of PCR containing 20 µM Cy3 (indocarbocyamine)-dUTP (Amersham Biosciences, Freiburg, Germany) and80 µM dTTP. PCR products were purified using a PCRpurification kit (Qiagen, Hilden, Germany) according to themanufacturer’s instructions.Preparation of arraysFor each SNP, 24 different 22-meric oligonucleotides weredesigned: 12 for the forward-strand sequence and 12 for thereverse-strand sequence. All four bases were representedat the polymorphic site, which was at position 11, 14 or 17from the 5′-end (Figure 1). Each oligonucleotide probe (10–20 µM solution) was spotted in triplicate/slide using thespotting device BioGrid (BioRobotics, Oxford, U.K.).The probes were covalently attached to the glass slides(Quantifoil, Jena, Germany) following the manufacturer’sinstructions. The surfaces of the slides were either epoxy-modified or aldehyde-modified. The probe-loaded slideswere blocked directly before hybridization following themanufacturer’s (Quantifoil) instructions.The ASOs had C6 linkers at their 5′-ends and wereeither SH- or NH2-modified, and were purchased from Inter-activa Biotechnologie GmbH (Ulm, Germany).Hybridization and washing of arraysLabelled PCR product (20 µl) from the targeted locus wasmixed with 180 µl of hybridization buffer [10 mM Tris/HCl,pH 7.5, 1 mM EDTA, 0.6 M NaCl, 1 × Denhardt’s solution(0.02% Ficoll 400, 0.02% polyvinylpyrrolidone and 0.02%BSA), 0.1 mg/ml herring sperm DNA and 0–50% form-amide]. Different formamide concentrations (0, 10, 20, 30,40 or 50%) were tested to determine the most appropriatestringency conditions. The hybridization cocktail was de-natured at 95 ◦C for 5 min, cooled on ice, applied to the arrayusing a hybridization frame (GeneScan, Freiburg, Germany)and incubated at 42 ◦C for 4 h. Subsequently, the slide waswashed for 10 min with 10 mM Tris (pH 7.5) and 39 mMNaCl at 32 ◦C, and then for 10 min with 10 mM Tris (pH 7.5)and 19.5 mM NaCl at 32 ◦C.Microarray reading and data analysisArrays were scanned on the GenePixTM 4000B scanner(Axon Instruments, Foster City, CA, U.S.A.) and imageswere analysed using the software GenePix TM Pro (AxonInstruments).Results and discussionThe ASO hybridization on glass slides required several opti-mization steps [14]. Epoxy- or aldehyde-modified glass sur-faces were tested with the same ASOs and hybridizationconditions. The signal intensities and signal-to-noise ratioswere comparable for both types of slides (results notshown). However, the use of alternative 5′-modificationsof ASOs affected the hybridization results substantially. 5′-SH-modified oligomers produced up to 35.4-fold strongersignals when compared with the same oligonucleotideshaving an NH2-modification. No difference in the signal-to-noise ratio was detectable (results not shown).Owing to the difficulty in predicting hybridization con-ditions and sequences of ASOs that will allow optimal allelediscrimination, these parameters had to be found empirically[10]. To select the appropriate oligonucleotide sequence,the strategy of probe redundancy [29] was chosen. Panelsof ASO probes containing the polymorphic site at variouspositions and querying the forward as well as the reverse-strand sequences were used for each SNP (Figure 1). In con-trast with ASO probes containing the polymorphic site atnucleotide position 17 from the 5′-end, ASOs with the allele-specific nucleotide at position 11 or 14 from the 5′-endC© 2005 Portland Press LtdQuantitative genotyping of SNPs using DNA-microarrays 95Figure 2 Quantitative genotyping results of four different genotypes of theA ↔ T-SNP BA213c14t7/403Results are shown for the optimized hybridization condition and ASO probesthat allowed the best discrimination between the alleles (hybridization buffercontaining 50% formamide; antisense ASO with polymorphic site at nucleotideposition 15 from the 5′-end). The heights of the histograms correspond to thenormalized fluorescence intensities of the spots. S.D. values were calculatedon the basis of signal intensities of the three replicas spotted for each ASOprobe. The allelic nucleotides are highlighted in black and grey respectively. Thegenotype T/T/T/T was not available.were both appropriate to discriminate between differentalleles. Sense and antisense oligonucleotides were tested todetermine the probe orientation that allowed better allelediscrimination. For the SNP locus BA213c14t7/403, signalsof the same quality were obtained with both sense andantisense strands, whereas, in the case of BA213c14t3/268 + 269, allele discrimination was only possible with theantisense ASOs (results not shown).The hybridization conditions appeared to be the mostimportant parameter requiring optimization. Several form-amide concentrations in the hybridization buffer were testedto achieve a strong and specific hybridization signal with alow background. It turned out that each SNP locus requiredspecific stringency conditions for optimal results. The besthybridization results were obtained using 10% (v/v) form-amide for BA44a10t7/174, 30% formamide for BA213c14t3/268 + 269 and 50% formamide for BA213c14t7/403. Therequirement to establish specific hybridization conditionsfor each SNP has been considered as a major drawback ofASO hybridization on microarrays [14].For SNP loci BA213c14t3/268 + 269 and BA213c14t7/403 the signal-to-noise ratio was sufficiently low for geno-typing the targeted polymorphisms. For SNP BA213c14t7/403, all four different genotypes, which were available,could be quantitatively discriminated (Figure 2). In case ofBA213c14t3/268 + 269, the expected hybridization signalsfor the two homozygous genotypes were obtained, but thequantitative accuracy failed to distinguish the different allelicratios of the three heterozygous genotypes (results notshown).For SNP BA44a10t7/174, the conditions used were notstringent enough to achieve a reliable genotyping (resultsnot shown). The signals obtained from the negative controls(ASO probes representing non-existing allelic bases) didnot allow robust genotyping. For this marker it seemsnecessary to improve other hybridization conditions suchas temperature and, possibly, primer design.In conclusion, our results suggest that it is possible toperform quantitative genotyping of SNPs using allele-specifichybridization on microarrays. Individual setting of speci-fic hybridization conditions for each SNP and selection ofappropriate material are two prerequisites to obtain reliablegenotyping results. To our knowledge, this is the firstattempt to show the feasibility of the method for quantitativegenotyping of SNPs. A comparison of advantages of thismethod with other techniques will only be possible whenmore examples will be available. Most of the methods testedso far for quantitative genotyping of SNPs [25] inherit theirdrawbacks. Therefore the procedure described here re-mains a promising alternative particularly for simultaneousanalysis of a large number of SNPs.AcknowledgmentsThis work was funded by BMBF (Bundesministerium fu¨rBilding und Forschung) grant 0312290A [GABI (GenomeAnalysis of the Plant Biological System)]. We thank SAKA-RAGIS Pflanzenzucht GbR for providing the plants.References1 Twyman, R. M. and Primrose, S. B. (2003) Pharmacogenomics4, 67–792 Collins, F. S., Guyer, M. S. and Chakravarti, A. (1997) Science278, 1580–15813 The International SNP Map Working Group (2001) Nature(London) 409, 928–9334 Reference deleted5 Long, A. D., Lyman, R. F., Langley, C. H. and Mackay, T. F. C.(1998) Genetics 149, 999–10176 Fan, J.-B., Chen, X., Halushka, M. K., Berno, A., Huang, X.,Ryder, T., Lipshutz, R. J., Lockhart, D. J. and Chakravarti, A.(2000) Genet. Res. 10, 853–8607 Buckler, E. S., Thornsberry, J. M. and Kresovich, S. (2001)Genet. Res. 77, 213–2188 Rickert, A. M., Jeong, H. 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Quantitative genotyping of single-nucleotide polymorphisms by allele-specific oligonucleotide hybridization on DNA microarrays

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