Single Nucleotide Polymorphism Analysis by ZipCode-Tagged Microspheres

Single Nucleotide Polymorphism Analysis by ZipCode-Tagged Microspheres

With the completion of a representative human genomic sequence, emphasis is increasingly shifting toward rapid, cost-effective, high-throughput analysis of individual sequence variation. Although a portion of this variation occurs as nucleotide insertions and deletions, substitutions in the form of single nucleotide polymorphisms (SNPs) are the most common. The total number of SNPs in the human genome may exceed 10 million, if those occurring in only 1% of the population are included (Kruglyak and Nickerson, 2001). High-density analysis of SNPs may uncover a correlation between specific genotypic patterns and disease states (Irizarry, 2001).

Because of this potential payoff, much effort has been focused on the design of SNP analysis platforms and techniques (Syvanen, 2001). Microsphere-based platforms offer a high level of multiplexing capability and provide an "open" system in which reactions can be modified easily by combining different sets of reagent-bearing microspheres (Fulton et al., 1997; Han et al., 2001; Kettman et al., 1998; Oliphant et al., 2002). A plethora of genotyping techniques harness the ability of DNA polymerases to perform high-fidelity extension of a primer along a complementary template (Cai et al., 2000; Chen et al., 2000; Syvanen, 1999; Taylor et al., 2001; Ye et al., 2001).

The genotyping technique detailed here employs a bifunctional capture probe possessing a 3' end complementary to the genomic target DNA and a 5' end complementary to an oligonucleotide-covered microsphere. DNA polymerase extends the capture probe with a labeled dideoxynucleotide complementary to the polymorphic base. Reaction products are afterward sequestered onto microspheres and analyzed with a benchtop fluorimeter.

N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide is from Pierce Biotechnology, Inc. (EDC, Cat. No. 22980). Tween 20 (Cat. No. P7949), 2-(N-morpholino) ethanesulfonic acid (MES, Cat. No. M2933), and 5 M NaCl (Cat. No. S5150) are from Sigma-Aldrich Corp. A 10% sodium dodecyl sulfate solution (SDS, Cat. No. 15553027), 20x SSC (Cat. No. 15557044), 1M Tris-HCl, pH 8.0 (Cat. No. 15568025), 0.5 M EDTA (Cat. No. 15575020), and distilled water (Cat. No. 15230162) are from Invitrogen Corp. Unlabeled ddNTPs are ordered as a set of four separate 5mM dideoxynucleotides from Amersham Biosciences Corp. (Cat. No. 27-2045-01), whereas unlabeled dNTPs are purchased as a blend of all four deoxynucleotides from Applied Biosystems (Cat. No. N808-0260). Biotin-11- ddATP (Cat. No. NEL548), biotin-11-ddCTP (Cat. No. NEL546), biotin-11-ddGTP (Cat. No. NEL549), and biotin-11-ddUTP (Cat. No. NEL547) are purchased as 1mM solutions from Perkin-Elmer Life Sciences. Streptavidin, R-phycoerythrin conjugate (1mg/ml) comes from Molecular Probes, Inc. (SA-PE, Cat. No. S-866).

AmpliTaq Gold DNA polymerase (Cat. No. 4311816) with accompanying GeneAmp 10x PCR Gold buffer and 25mM MgCl2 are from Applied Biosystems. Shrimp alkaline phosphatase (SAP, Cat. No. E70092Z), exonuclease I (Cat. No. E70073Z), and Thermo Sequenase DNA polymerase (Cat. No. E79000Y) with included 10x Thermo Sequenase reaction buffer are from Amersham Biosciences Corp.

Biotinylated and unmodified DNA oligonucleotides are synthesized by BioSource International. Aminomodified oligonucleotides are from Oligos Etc., Inc. Human genomic DNA from CEPH reference families is obtained from Coriell Cell Repositories.

The 96-well microtiter plates (Cat. No. AB-0800) and adhesive plate film (Cat. No. AB-0558) are from Marsh Bioproducts. A VWR Model 75D sonic water bath from VWR Scientific Products is used to sonicate microspheres. A Sorvall RT6000D tabletop centrifuge from Kendro Laboratory Products is used to spin microtiter plates. Sheath fluid (Cat. No. 40-50000), carboxylated microspheres (Cat. Nos. L100-Cl01-01 through L100- C200-01), and the Luminex 100 system are from Luminex Corporation. The RapidPlate 96/384 microplate pipetting workstation is from Zymark Corporation. The PTC-100 thermocyclers are from MJ Research.

A. Design and Preparation of Oligonucleotides
  1. Design and synthesize forward and reverse PCR primers for amplification of each genomic polymorphism to be assayed. Primers should have a melting temperature (Tm) between 52 and 64°C. Designing all PCR primers to share a common Tm simplifies reaction setup and aids multiplex PCR strategies. Primer positions should be selected so that amplicons are as small as possible, ideally less than 100bp, with the polymorphic base positioned centrally. Make 100 µM stocks in distilled water.
  2. Synthesize a complementary ZipCode (cZip- Code) oligonucleotide for each microsphere type being used (up to 100 types). The sequences of a large set of compatible ZipCodes have been published (Iannone et al., 2000). Oligonucleotides should have a 5' amino group, followed by a 15 to 18-atom spacer. Good results are achieved with use of Uni-Link reagent (amino group plus 6-carbon spacer) followed by a 9- atom spacer. The spacer is followed by a 20 nucleotide tag sequence (LUC) common to all cZipCodes. Incorporation of this tag allows multiplexed quality assurance assays to be performed after microspheres are covalently coupled with oligonucleotides. The LUC tag is an arbitrarily selected portion of the luciferase reporter gene that does not share homology with human genomic targets. The LUC tag is followed by the 25 nucleotides complementary to the ZipCode. The Tm of the cZipCode, excluding the LUC tag, is between 61 and 66°C. The following cZipCode example has the LUC tag underlined: 5'-mamino-15 carbon-CAG GCC AAG TAA CTT CTT CGC CGT ACC CTT CCG CTG GAG ATT TAC-3'. Make 1mM stocks in distilled water.
  3. Synthesize a biotinylated anti-LUC oligonucleotide with the sequence 5'-CGA AGA AGT TAC TTG GCC TG-3'. The 20mer is biotinylated at the 5' end and has a Tm of 52°C. Make 10µM stocks in distilled water.
  4. Design and synthesize a capture probe for each polymorphism to be assayed. Probes have a 25- nucleotide ZipCode at the 5' end and a genomic targetspecific 3' end, terminating immediately before the polymorphic base. The Tm of the genomic targetspecific portion should range between 51 and 56°C. The sequence of the positive control capture probe (step 5) serves as an example of these design rules. Make 100µM stocks in distilled water.
  5. Synthesize a positive control capture probe with the sequence 5'-GTA AAT CTC CAG CGG AAG GGT ACG GAT CGG CGA TGC ACT TGG ATT-3'. The ZipCode (Tm = 61°C) is located at the 5' end and the target-specific sequence (Tin = 52°C) is underlined. Make 10µM stocks in distilled water.
  6. Synthesize a positive control target oligonucleotide with the degenerate sequence 5'-AAC CAG CGG GGC AAC CAA CNA ATC CAA GTG CAT CGC CGA T-3'. The sequence to which the positive control capture probe anneals is underlined. The incorporation of a fourfold degenerate base (N) allows use of a single positive control target, independent of the SNP allele being assayed. Make 10µM stocks in distilled water.

B. Covalent Coupling of Oligonucleotides to Carboxylated Microspheres
  1. 0.1M MES, pH 4.0: To make 50ml, dissolve 0.976 g MES in 40ml distilled water. Adjust pH to 4.0 and add distilled water up to 50 ml. Store at room temperature for months.
  2. 30mg/ml EDC: Weigh 15 mg EDC directly into a microfuge tube, avoiding any clumps in the stock bottle. Resuspend in 500 µl distilled water immediately before addition to microspheres. Solid EDC is highly hygroscopic and should be replaced each month. For large-scale couplings, always use a new bottle of EDC to minimize risk of failure in the coupling reaction. Do not prepare this solution in advance.
  3. 0.1% SDS: To make 50ml, add 500µl 10% SDS stock to 49.5 ml distilled water. Store at room temperature for months.
  4. 0.02% Tween 20: To make 50ml, add 10µl Tween 20 to 49.99ml distilled water. Store at room temperature for months.
  5. 1× TE, pH 8.0 (10mM Tris-HCl, pH 8, 1mM EDTA): To make 500ml, combine 5ml 1M Tris-HCl, pH 8, 1ml 0.5M EDTA, pH 8, and 494ml distilled water. Store at room temperature for months.

The following protocol has been used for coupling reactions of various sizes, ranging from 625,000 microspheres (50µl of stock) to 100 million microspheres (8ml of stock). The only reagent volume adjustment required with increasing microsphere number is a doubling of MES resuspension volumes to 100 µl (step 4) when more than 1ml of microspheres is coupled.
  1. Remove desiccant jar containing solid EDC from freezer and warm to room temperature before opening.
  2. Pellet 1ml of microspheres (12.5 million) by centrifugation in a benchtop microfuge for 5 min at full speed.
  3. Carefully remove supernatant by pipette, leaving microsphere pellet completely dry.
  4. Add 50µl 0.1M MES, pH 4.0, and resuspend microspheres.
  5. Add 1.5µl of the appropriate 1mM aminomodified cZipCode oligonucleotide dissolved in distilled water.
  6. Prepare a fresh 30-mg/ml solution of EDC in distilled water.
  7. Immediately after solvation of the EDC, add 10µl EDC solution to microspheres. Vortex and sonicate immediately.
  8. Incubate at room temperature in the dark for 30min. Vortex and sonicate every 10min during this incubation step.
  9. Repeat steps 6-8.
  10. After the second 30-min incubation, add 800µl 0.1% SDS, vortex, pellet microspheres, and remove supernatant.
  11. Add 800 µl 0.02% Tween 20 to microspheres, vortex, pellet microspheres, and remove supernatant.
  12. Resuspend microspheres in 1ml 1× TE, pH 8.0, to make stocks of approximately 10,000 microspheres/µl. Keep individual microsphere types segregated until a quality control assay has been performed to assess coupling effectiveness for each microsphere.
  13. Wrap tubes of microspheres in aluminum foil and store at 4°C.

C. Multiplexed Quality Control Assay for Newly Coupled Microspheres
  1. Wash solution (1× SSC/0.02% Tween 20): To make 500 ml, combine 25 ml 20x SSC, 100 µl Tween 20, and 475ml distilled water. Store at room temperature for months.
  2. Staining solution (8.33µg/ml SA-PE): To make 50ml, mix 417µl SA-PE with 49.6 ml wash solution. Store wrapped in aluminum foil at 4°C for several months.

  1. Mix together 1µl of each microsphere type to be tested (up to a maximum of 20 microsphere types). Supplement with distilled water if final volume is less than 20 µl to create a final concentration of 500 microspheres of each type/µl.
  2. If a previously tested microsphere is available that successfully passed the quality control assay and that is a different type from the multiplexed microspheres assembled in step 1, that microsphere can be used as an internal positive control. Dilute 1 µl of that microsphere with 19µl of distilled water to create a solution of 500 microspheres/µl.
  3. Combine in a single well of a 96-well plate the following: 2µl of untested multiplexed microspheres (from step 1), 2µl of a previously tested microsphere (from step 2), 2µl 5M NaCl, 0.5µl of 10µM biotinanti- LUC oligonucleotide for each microsphere type (maximum of 10.5µl of oligonucleotide for 21 microsphere types), and distilled water up to 20µl final volume.
  4. Set up a second reaction that is similar to step 3, but omit the biotin-anti-LUC oligonucleotide. This reaction serves as a negative control.
  5. Seal plate and incubate at 37°C for 1h.
  6. Add 120µl wash solution to each well. Pellet microspheres by centrifuging plate at 2000rpm for 5 min. Carefully remove supernatant.
  7. Resuspend microspheres in 65µl staining solution and incubate at room temperature in the dark for 30 min.
  8. Place plate in 1×100 and read 50µl from each well. Successfully coupled microspheres will generally have mean fluorescent intensity (MFI) values of several thousand units. Microspheres with MFI values below 1000 should be recoupled.

D. Amplification of Genomic Targets and Amplicon Cleanup
  1. 1.3x PCR mix (261 µM dATP, 261µM dCTP, 261 µM dGTP, 261µM dTTP, 1.3x GeneAmp PCR Gold buffer (19.5mM Tris-HCl, pH 8.0, 65mM KCl), 3.91mM MgCl2): To make 1265 µl (enough for one 96-well plate), add 770 µl distilled water, 132µl of 10mM dNTP blend (2.5mM of each dNTP), 198µl 25mM MgCl2, and 165 µl GeneAmp 10x PCR Gold buffer. This solution can be made in large batches and stored in single-thaw aliquots at -20°C.
  2. PCR primer mix (1µM each oligonucleotide primer): To make 330µl (enough for one 96-well plate), combine 3.3µl of each 100µM forward primer, 3.3µl of each 100µM reverse primer, and dilute to 330µl with distilled water. Carefully designed primer pairs that share a common Tm and that lack cross-complementarity may be multiplexed to robustly amplify up to 10 small amplicons simultaneously.
  3. PCR master mix (200µM dATP, 200µM dCTP, 200µM dGTP, 200µM dTTP, 3.0mM MgCl2 1× GeneAmp PCR Gold buffer (15mM Tris-HCl, pH 8.0, 50mM KCl), 200nM each PCR primer, 166.7 units/ml AmpliTaq Gold DNA polymerase): To make 1650 µl (enough for one 96- well plate), combine 1265 µl 1.3x PCR mix, 330µl PCR primer mix, and 55µl of 5U/µl AmpliTaq Gold DNA polymerase. For best results, use master mix immediately.
  4. SAP/Exo digestion mix (1× GeneAmp PCR Gold buffer, 0.4U/µl SAP, 0.8U/µl exonuclease I): To make 550µl (enough for one 96-well plate containing 15-µl PCR reaction volumes), combine 55µl GeneAmp 10x PCR Gold buffer, 220µl SAP, 44µl exonuclease I, and 231µl distilled water. For best results, prepare just prior to use.

  1. Dispense 20 ng genomic DNA into each well of a 96- well plate. Heat to dryness by placing plates into a 55°C oven for 2h. Dried plates may be safely stored at room temperature for up to 1 month.
  2. Add 15µl PCR master mix to each well of dried genomic DNA plate. 3. Seal plate and spin in centrifuge at 2000rpm for 20s.
  3. Place plate in thermocycler and heat at 95°C for 10min, followed by 40 cycles of 94°C for 30s, 60°C for 30s, and 72°C for 30s, finishing with 5min at 72°C and 4°C hold.
  4. If multiplex PCR was not performed, combine equal volumes of all PCR amplicons generated across multiple plates into one common plate for multiplex cleanup. It is not necessary to combine the entire volume from the individual PCR reactions, but the total combined volume must be at least 15 µl per well.
  5. For every 15µl of PCR reaction volume per well, add 5 µl SAP/Exo digestion mix.
  6. Mix thoroughly.
  7. Incubate in thermocycler at 37°C for 30min, followed by denaturation of enzymes at 80°C for 15 min and 4°C hold.
  8. Plates may be safely stored at 4°C overnight or at -20°C for several days.

E. Polymerase Assay
  1. 20× positive control (250nM positive control capture oligonucleotide, 250nM positive control target oligonucleotide): To make 550µl, mix 13.75µl 10µM positive control capture oligonucleotide, 13.75 µl 10 µM positive control target oligonucleotide and 522.5µl distilled water. Store at 4°C for up to I week or store frozen for several months, but avoid refreezing.
  2. 20× capture probe multiplex (500 nM solution of each capture probe): To make 550 µl, combine 2.75 µl of each 100 µM capture probe stock and add distilled water up to 550µl. Store at 4°C for up to 1 week or store frozen for several months, but avoid refreezing.
  3. 5x capture probe/positive control mix: To make 2200µl (typically enough for four 96-well plates of genotyping reactions), combine 550µl of 20x positive control, 550µl of 20x capture probe multiplex, and 1100µl distilled water. Store at 4°C for up to I week or store frozen for several months, but avoid refreezing.
  4. 1000× minus-A solution (1 mM ddCTP, 1mM ddGTP, 1mM ddTTP): To make 20µl (enough for nine plates of reactions), combine 4µl 5mM ddCTP, 4µl 5 mM ddGTP, 4 µl 5 mM ddTTP, and 8 µl distilled water. Store at 4°C for several days or store frozen for several months, but avoid multiple freeze-thaw cycles. Keep in mind that this solution is one specific example of the four solutions required to analyze all of the four possible alleles (A, C, G, and T). To assay an A/G biallelic SNP, you must also prepare a 1000x minus-G solution by substitution of 1mM ddGTP with 1mM ddATP.
  5. 2.5× A mix (2.5× Thermo Sequenase reaction buffer, 2.5µM ddCTP, 2.5µM ddGTP, 2.51dVl ddTTP, 2.5µM biotin-11-ddATP): To make 880µl (enough for one 96- well plate of reactions), combine 220µl 10x Thermo Sequenase buffer, 2.2 µl 1000x minus-A solution, 2.2 µl 1mM biotin-11-ddATP, and 655.6µl distilled water. Store at 4°C for several days or store frozen for several months, but avoid multiple freeze-thaw cycles. Keep in mind that this solution is one specific example of the four solutions required to analyze all of the four possible alleles (A, C, G, and T). To assay an A/G biallelic SNP, you must also prepare a 2.5x G mix by substitution of 2.5µM ddGTP with 2.5 µM ddATP and replacement of 2.5µM biotin-11-ddATP with 2.5µM biotin- 11-ddGTP.
  6. 3× microsphere multiplex (100 microspheres of each type/µl, 1.5M NaCl, 40mM EDTA): To make 2200µl (enough for two 96-well plates of reactions), combine 22µl of each type of oligonucleotide-coupled microsphere (10,000/µl), 660 µl 5M NaCl, 176 µl 0.5M EDTA, and distilled water up to 2200µl. If more than 62 microsphere types are being multiplexed, then the total volume will exceed 2.2ml. In this case, combine all microsphere types, pellet the microspheres in a centrifuge, and carefully remove a sufficient volume of supernatant before the addition of NaCl and EDTA so that the final volume will equal 2200µl. If protected from light by wrapping the tube in aluminum foil, this solution can be kept for over 1 year at 4°C
  7. Wash solution: See Section III,C, solution 1.
  8. Staining solution: See Section III,C, Solution 2.

  1. Retrieve plate of multiplexed, digested PCR amplicons from Section III,D, step 9. For every 20µl of volume per well, add 10µl of 5x capture probe/ positive control mix.
  2. From this plate containing PCR amplicons, capture probes and positive control, split out two allele-specific reaction plates by placing 12 µl/well into each of two 96-well plates.
  3. Add 10µl of Thermo Sequenase DNA polymerase to each 880-µl aliquot of 2.5x allele-specific mix. For example, add 10µl Thermo Sequenase DNA polymerase to 880µl 2.5x A mix and 10µl Thermo Sequenase DNA polymerase to 880µl 2.5x G mix.
  4. Complete each allele-specific reaction plate by the addition of 8.1µl/well of 2.5x A/enzyme mix to one plate and 8.1µl/well of 2.5x G/enzyme mix to second plate. Each well contains a 20.1-µl reaction with 1× Thermo Sequenase reaction buffer, 1µM each of three unlabeled ddNTPs, 1µM of the fourth biotinlabeled ddNTP, multiplexed PCR products, multiplexed capture probes at 25 nM each, 12.5 nM positive control capture probe, 12.5 nM positive control target oligonucleotide, and 145 units/ml Thermo Sequenase DNA polymerase.
  5. Seal plates, centrifuge briefly, and place into thermocyclers for a 2-min denaturation at 96°C followed by 40 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 30s, finally holding at 25°C.
  6. Remove plate sealers and add 10µl/well of 3x microsphere multiplex to each plate. Mix well, reseal each plate, and place into thermocyclers. Incubate at 45°C for 1h to sequester capture probes onto the microsphere surfaces.
  7. Remove plate sealers and add 150µl/well of wash solution. Centrifuge plates for 5 min at 2000rpm to pellet microspheres. Carefully remove supernatant containing excess biotin-labeled ddNTPs using a RapidPlate 96/384 microplate pipetting workstation.
  8. Resuspend each microsphere pellet in 65µl of staining solution. Incubate at room temperature in the dark for 30 min.
  9. Plates can be analyzed in the Luminex 100 system without further processing. Configure instrument settings to inject 50µl of sample from each well and analyze at least 30 microspheres of each type per well (30 events).

Using the protocols described in this article, it is possible to routinely perform 50plex genotyping reactions, generating up to 4800 genotypes per pair of 96- well allele-specific plates.

  1. Failure to covalently couple oligonucleotides onto the carboxylated microspheres can frequently be traced to the EDC reagent. Dry EDC is a fine powder. Formation of clumps is a sign of water absorption and will result in poor coupling.
  2. Another potential cause of failure in the coupling reaction is use of a poorly synthesized oligonucleotide. Always perform a small test coupling when a new oligonucleotide is synthesized. After an oligonucleotide has been demonstrated to couple effectively, freeze away small aliquots at -70°C for single-thaw use.
  3. Microspheres can be photobleached by ambient laboratory light, causing them to lose their spectral identities. Because this is a cumulative process, longterm microsphere stocks, such as bulk carboxylated microspheres and oligonucleotide-coupled stocks, should be rigorously protected by wrapping in aluminum foil. Coupling reactions in progress should always be placed inside a bench drawer or shaded under a sheet of aluminum foil. Likewise, polymerase reaction plates should not be left unprotected on the benchtop while waiting for availability of a centrifuge or Luminex 100.
  4. Occasionally, a capture probe will consistently produce reaction products that display low signal strength or high backgrounds, resulting in poor signalto- noise ratios. It is sometimes possible to correct this problem by redesigning the capture probe from the opposite genomic strand.
  5. Effective removal of unincorporated biotinylated dideoxynucleotides during the wash step can be critical to maximize signal-to-noise ratios. This is especially true for SNPs with weaker signal strength. Remove the largest practical volume of supernatant from the microsphere pellet without risking disturbance of the pellet and resulting loss of microspheres.

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