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Automation and Analytical Techniques |
Departments of1
Chemical and Biochemical Engineering and 2
Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ.
3 School of Public Health/Environmental and Occupational Health Sciences Institute, University of Medicine and Dentistry of New Jersey, Piscataway, NJ.
aAddress correspondence to this author at: Biomedical Engineering, Rutgers University, 599 Taylor Rd., Rm. 207, Piscataway, NJ 08854. Fax 732-445-3753; e-mail sobinkim{at}rci.rutgers.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used the previously developed solid-phase capture–single-base extension (SPC-SBE) approach for concurrent analysis of 40 single-nucleotide polymorphisms (SNPs) of CYP2C9 and 50 SNPs of CYP2A13, both genes belonging to the CYP450 family. Desired SNP-containing regions for each gene were amplified in a single-step multiplex PCR. We designed a library of primers to anneal immediately upstream of the selected SNPs and extended it with biotinylated terminators using PCR products as templates. Biotinylated extension products were isolated by affinity purification and analyzed with MALDI-TOF mass spectrometry to determine SNP genotypes.
Results: We analyzed 11 samples for CYP2C9 and 14 samples for CYP2A13 with unambiguous detection of SNPs in all samples. Many samples showed a high occurrence of heterozygotes for both genes, with as many as 10 of 50 SNPs appearing as heterozygotes in 1 sample genotyped for CYP2A13.
Conclusions: The SPC-SBE method provides an efficient means for genotyping SNPs from the CYP450 family. This approach is suitable for automation and can be extended to other genotyping applications.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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90% of genetic variations and are commonly used as markers in association studies for correlating genotypes and phenotypes (1)(2). Many techniques have been developed for SNP genotyping; they typically involve the generation and detection of allele-specific products (3)(4)(5)(6). MALDI-TOF MS has emerged as a high-throughput detection platform for SNP genotyping because of its short analysis time and capacity for quantitation (7)(8)(9)(10). Different approaches have been coupled with MALDI-TOF MS for this purpose, including hybridization, cleavage, ligation, and primer extension (7). Of these, methods based on primer extension are most widely used because of their high accuracy and scope for analyzing multiple SNPs simultaneously (7)(11).
A new MALDI-TOF MS technique based on primer extension, solid-phase capture–single-base extension (SPC-SBE; Fig. 1
), can analyze larger numbers of nucleotide variations (12). Briefly, it involves PCR amplification of desired genomic regions followed by inactivation of dNTPs and PCR primers. The PCR products are then used as templates for extension of SBE primers with dideoxyribonucleotide triphosphates (ddNTPs) attached to a biotin moiety. Extension products are terminated with biotin-ddNTPs and isolated by affinity purification on a streptavidin-coated solid surface. They are then desalted and analyzed with MALDI-TOF MS to yield a spectrum containing peaks of extension products only. Genotypes are inferred by comparing masses of peaks from the spectrum with precalculated masses of expected extension products. Elimination of peaks corresponding to unextended SBE primers from the resulting mass spectrum allows detection of much larger numbers of extension products, leading to higher levels of multiplexing. The feasibility of multiplex SNP genotyping using SPC-SBE has been demonstrated by simultaneous screening of 30 point mutations in the p53 gene (13). We report here SPC-SBE assays for multiplex genotyping of SNPs from 2 genes of the cytochrome P450 (CYP450) family.
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CYP450 isoenzymes are involved in the phase 1–dependent metabolism of 70%–80% of all therapeutically important drugs (14). CYP2C92
is involved in the metabolism of 10%–15% of these drugs, including tolbutamide, S-warfarin, and many nonsteroidal antiinflammatory drugs (15)(16). The gene is highly polymorphic, with approximately 290 SNPs in the National Center for Biotechnology Information (NCBI) dbSNP database (http://www.ncbi.nlm.nih.gov/SNP) and 30 alleles in the Human CYP allele database (http://www.cypalleles.ki.se) (17)(18). Many of these alleles result in altered catalytic activity of the corresponding protein for different substrates (16). CYP2A13 is one of the less well characterized members of the CYP450 family and was initially thought to be nonfunctional. Only recently has its role been demonstrated in the activation of 2 important environmental carcinogens, the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and aflatoxin B1 (19)(20). Fewer than 70 SNPs of the gene have been reported in the NCBI dbSNP and Human CYP allele databases combined, and only a few of those have been functionally characterized. Since CYP2A13 was characterized, there have been few studies involving SNPs of the gene using PCR single-strand conformation polymorphism or direct sequencing for SNP analysis (21). Because of wide applications in pharmacogenomics and environmental cancer studies, there is a need for genotyping SNPs of CYP2C9 and CYP2A13. Here we demonstrate simultaneous genotyping of 40 SNPs of CYP2C9 and 50 SNPs of CYP2A13 in separate assays using the SPC-SBE approach.
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Materials and Methods |
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snp selection and sbe primer design
We selected 40 exonic and intronic SNPs in the vicinity of exons 3, 6, and 7 for CYP2C9, and 50 SNPs spanning the entire gene including all exonic SNPs for CYP2A13, from the NCBI dbSNP and Human CYP allele databases. For each SNP, we designed SBE primers with sufficient mass difference between successive primers to accurately perform multiplex genotyping and avoid overlap between double-charged ions of larger extension products and single-charged ions of smaller extension products (see Tables 1 and 2 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue5). For SNPs on adjacent bases, we selected a reverse primer that anneals to the complementary strand to detect the latter SNP, and also selected reverse primers for some other SNPs as indicated (see Table 2 in the online Data Supplement).
pcr amplification
We designed PCR primers to amplify genomic regions containing the selected SNPs for both genes (see Tables 3 and 4 in the online Data Supplement) using reference sequences from GenBank (accession nos. AY341248 for CYP2C9 and NG_000008 for CYP2A13) (22). We selected 3 pairs of primers for CYP2C9 to amplify desired SNP-containing regions and 7 pairs for CYP2A13 to cover all selected SNPs and verified the uniqueness of primer sequences using NCBI BLAST to prevent nonspecific amplification of other members of the CYP450 family (23). We obtained all PCR primers from Integrated DNA Technology and performed all PCRs on a PTC-200 thermocycler (MJ Research). For CYP2C9, we mixed primers (12 pmol F1 and R1, 12 pmol F3 and R3, and 15 pmol F2 and R2) with 500 ng genomic DNA, 2.5 mmol/L dNTPs, 4 units Ex Taq enzyme (Takara Mirus Bio), and 1x Ex Taq buffer in a 65-µL reaction volume. PCR conditions were as follows: 94 °C hot start for 2 min, followed by 40 cycles of 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 120 s, with final extension at 72 °C for 10 min. For CYP2A13, we mixed primers (5 pmol F1 and R1, 5 pmol F2 and R2, 5 pmol F4 and R4, 5 pmol F5 and R5, 7.5 pmol F3 and R3, 7.5 pmol F6 and R6, and 7.5 pmol F7 and R7) with 500 ng genomic DNA, 2.5 mmol/L dNTPs, 3 units Ex Taq enzyme, and 1x GC buffer II in a 35-µL reaction volume. PCR conditions were as follows: 94 °C hot start for 2 min, followed by 40 cycles of 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 70 s, with a final extension at 72 °C for 10 min. PCR products (3 µL) were run on a 1.2% agarose gel with 1x Tris-acetate-EDTA buffer for visualization. Next, PCR products (50 µL) were incubated with 5 units exonuclease I and 5 units shrimp alkaline phosphatase (USB Corp.) at 37 °C for 90 min to inactivate excess PCR primers and dNTPs, respectively, followed by enzyme deactivation at 94 °C for 15 min. We performed PCR amplification of individual regions using the same conditions with region-specific forward and reverse primers only.
sbe
We obtained all SBE primers from Midland Certified Reagent Co. For CYP2C9, we mixed 35 µL corresponding PCR cleanup products with SBE primers (see Table 1 in the online Data Supplement), 210 pmol biotin-11-ddA, 500 pmol biotin-11-ddC, 260 pmol biotin-11-ddG (PerkinElmer), 250 pmol biotin-16-ddU (Enzo Diagnostics), 10 units Thermo Sequenase enzyme (GE Healthcare), and 1x reaction buffer in a 75-µL reaction volume. Cycling conditions for SBE were as follows: 94 °C hot start for 2 min, followed by 40 cycles of 94 °C for 30 s and 58 °C for 30 s. For CYP2A13, we mixed 35 µL corresponding PCR cleanup products with SBE primers (see Table 2 in the online Data Supplement), 300 pmol biotin-11-ddA, 550 pmol biotin-11-ddC, 450 pmol biotin-11-ddG, 500 pmol biotin-16-ddU, 12 units Thermo Sequenase enzyme, and 1x reaction buffer in an 85-µL reaction volume. Cycling conditions for SBE were as follows: 94 °C hot start for 2 min, followed by 10 cycles of 94 °C for 30 s and 72 °C for 30 s, followed by 35 cycles of 94 °C for 30 s and 62 °C for 30 s.
spc
We separated the extension products from unextended SBE primers by use of streptavidin-coated magnetic beads (Seradyn) as follows. The bead solution (80 µL) was prewashed twice with 90 µL binding-washing buffer (1 mol/L ammonium chloride, 2x Tris-HCl EDTA, pH 7.3) and resuspended in 90 µL binding-washing buffer. We mixed the products from the SBE reaction with beads and incubated them for 1 h with constant vortex-mixing, then washed the beads twice with 180 µL binding-washing buffer and once with 180 µL of 0.1 mol/L triethylammonium acetate solution and 180 µL deionized water. The beads were then resuspended in 12 µL formamide and incubated at 94 °C for 7 min to denature the streptavidin-biotin complex. We magnetically separated formamide containing extension products from the beads and mixed them with 120 µL ethanol for overnight incubation. The solution was centrifuged at 20 817g for 45-min, followed by removal of supernatant and drying of formamide. The dried contents were then resuspended in 20 µL of 0.1 mol/L triethylammonium acetate solution for desalting using a reversed-phase ZipTip C18 column (Millipore).
maldi-tof ms analysis
The desalted DNA was dried and resuspended in 1 µL deionized water and 1 µL matrix and spotted on a 100-well stainless steel sample plate. Matrix consisted of 35.6 mg 3-hydroxypicolinic acid and 6.2 mg ammonium citrate dissolved in 800 µL of 50% acetonitrile solution (Sigma-Aldrich). We performed MALDI-TOF MS analysis in linear positive mode of a Voyager DE Pro instrument (Applied Biosystems) using 25 kV accelerating voltage, 94% grid voltage, 0.07% guide wire voltage, and a delay time of 350 ns. For each spectrum, 75 shots were taken, and 4 spectra were accumulated for each sample. We performed external calibration by use of 5 unextended SBE primers in the 5000 to 11 000 Da interval as mass calibrators and used Data Explorer software provided with the instrument for noise removal and smoothing on each accumulated spectrum.
direct sequencing
We performed bidirectional sequencing of individual regions with individual PCR products and corresponding forward or reverse PCR primer on an ABI 3730xl capillary gel sequencer using BigDye® Terminator v3.1 mix (Applied Biosystems).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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snp genotype determination by spc-sbe
We used PCR products as templates for simultaneous extension of corresponding SBE primers and isolated and analyzed extension products by MALDI-TOF MS to determine SNP genotypes. A representative mass spectrum for the analysis of 40 SNPs in CYP2C9 is shown in Fig. 2A
(also see Fig. 2A in the online Data Supplement). Genotypes of all 40 SNPs were unambiguously determined by comparing masses of peaks from the spectrum with expected masses (see Table 1 in the online Data Supplement). For example, in the case of Fig. 2A
, a peak is observed at a mass of 7199 Da. When compared with expected masses for CYP2C9, this corresponds to the A extension of primer for SNP 16, indicating that the SNP is homozygous for A. Similarly, 2 peaks are observed at 10 134 and 10 223 Da in the same spectrum. On comparing these masses with expected masses, both are seen to result from extension of primer for SNP 37, indicating that the SNP is a C/T heterozygote. For CYP2A13, 50 SNPs were analyzed simultaneously, and genotypes for all SNPs were inferred by comparing masses of extension products with expected masses (see Table 2 in the online Data Supplement). A representative spectrum is shown in Fig. 2B
, with strong spectral peaks observed even in the higher mass range (also see Fig. 2, B and C, in the online Data Supplement). SNP genotypes for all samples that were analyzed matched those that have been previously reported in the NCBI dbSNP and Human CYP allele databases.
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direct sequencing for validation of snp genotypes
For validating results obtained by SPC-SBE, we sequenced individual regions using capillary gel electrophoresis for 2 of the CYP2C9 cell line–based samples and for 4 of the cell line–based samples and 1 anonymous donor sample for CYP2A13. A perfect match was seen between genotypes deduced from SPC-SBE and those obtained by direct sequencing.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, resulting in accurate genotyping of SNPs. Also, minimum mass difference between heterozygotes increases to 16 Da for A/G heterozygote in SPC-SBE because of the use of biotinylated ddNTPs, compared with 9 Da for A/T heterozygote with regular ddNTPs. This leads to better resolution for heterozygotes and ensures their unambiguous detection, as demonstrated by the spectra in Fig. 2
. Fig. 2A
shows the detection of an A/T heterozygote in the largest extension product with good resolution over 10 000 Da. Fig. 2B
clearly shows an A/G heterozygote for extension of primer 30 with good resolution even at 9500 Da. A mass difference of more than 105 Da between successive primers ensures a minimum mass difference of 16 Da between all combinations of extension products. This allows detection of all 4 possible genotypes for each SNP, leading to identification of additional alleles that may not have been reported at a SNP site.
SPC-SBE has been used for simultaneous analysis of 30 point mutations in the p53 gene (13). A mutation is a single-base variation that is usually found to occur at a very low frequency, and hence different samples are expected to have similar spectra with little variation when detecting mutations. A SNP, by definition, must have a less frequent allele that occurs in >1% of the population. Consequently, for the case of SNP genotyping, there is much higher likelihood of obtaining varying genotypes in different samples with high occurrence of heterozygotes, making the analysis of SNPs more difficult than the analysis of point mutations—for example, Fig. 2A
shows a spectrum for 40-SNP genotyping with 3 heterozygotes; Fig. 2B
shows a spectrum for 50-SNP genotyping with 10 heterozygotes. We were able to deduce unambiguous SNP genotypes for all samples based on spectra obtained from the analysis of extension products for both CYP2C9 and CYP2A13. Simultaneous genotyping of 50 SNPs is the highest level of multiplexing reported so far using MALDI-TOF MS.
Previous studies with SNPs in CYP2A13 have focused on the discovery of SNPs in the gene, and to the best of our knowledge no assays with high levels of multiplexing have been applied for genotyping them. For CYP2C9, assays for multiplex genotyping of SNPs have been developed, focusing on genotyping exonic SNPs using hybridization with oligonucleotide array, pyrosequencing, and SNaPshot® approaches (24)(25)(26). Pyrosequencing is a novel approach that uses chemiluminescence detection but is currently limited in its capacity for genotyping multiple SNPs. The array-based hybridization approach for genotyping uses fluorescence detection but is better suited to studies that involve genotyping of large numbers of SNPs in a limited number of samples (6)(11). SNaPshot technology uses fluorescence detection and has been used for genotyping 3 SNPs of CYP2C9 (26). The approach uses SBE with fluorescently labeled ddNTPs followed by capillary gel electrophoresis for SNP genotyping and has been used for analysis of up to 35 SNPs simultaneously (27). In principle, the first step for SNP genotyping in the SNaPshot method, SBE for generation of allele-specific products, is the same as SPC-SBE. The main difference between the 2 techniques lies in the way the extension products are detected and used to infer SNP genotype. In SPC-SBE, SNP genotype is deduced by mass spectrometry using direct measurement of the mass-to-charge ratio, which is an intrinsic property for any molecule. Most other techniques like SNaPshot use indirect measures, such as fluorescence, for detection. Because mass spectrometry relies on an intrinsic property for detection, it has less likelihood of error compared with indirect measures. This is particularly true in the case of heterozygotes that may be relatively harder to detect using fluorescence, owing to overlapping peaks in the electropherogram (28). In contrast, a mass spectrum leads to unambiguous detection of a heterozygous SNP owing to the presence of multiple extension peaks.
In SPC-SBE, mass accuracy and mass resolution of spectral peaks are equally important in assigning SNP genotypes. To achieve good mass accuracy, we used 5 mass calibrators in the interval of 5000 to 11 000 Da for external calibration of our samples. This resulted in a difference of <3 Da between observed and expected masses for all peaks, allowing accurate calling of genotypes. Mass resolution is important in distinguishing closely spaced peaks and is especially important in identifying heterozygotes. With the SPC procedure, we were able to increase minimum mass difference between heterozygotes to 16 Da and enhance sample purity to reduce formation of adducts leading to accurate peak identification. For example, in the case of Fig. 2A
, 3 heterozygotes are observed with the following genotypes: A/G, C/T, and A/T. Of these, the C/T and A/T heterozygotes are easily distinguishable by mass differences of 89 and 66 Da between corresponding extension products, respectively. The A/G heterozygote is comparatively harder to distinguish, with its smaller mass difference, but a magnified view of the region (Fig. 2A
, inset) clearly shows peaks corresponding to both extension products.
In contrast to problems resulting from mass accuracy and resolution, errors inherent in the SBE-SPC procedure are impossible to distinguish except by comparative study with a different technique. False positives can arise because of the presence of an additional peak in the spectrum resulting from nonspecific annealing and extension of a primer. False negatives can arise because of insufficient capture of an extension product, resulting in the absence of its corresponding peak from the spectrum, which would lead to improper genotype calling. We analyzed 25 samples in this study, with confirmation of genotypes in 7 samples by direct sequencing; however, a much larger sample set would be required to adequately address the rate of false calls with the technique.
Further improvement in multiplexing levels using SPC-SBE are limited by inherent problems with analysis of larger-mass oligonucleotides using MALDI-TOF MS. Larger oligonucleotides are more susceptible to fragmentation during detection and give rise to peaks that are not well resolved, with low signal-to-noise ratio. As a result, it is difficult to use them for genotyping applications that require measurement of small mass differences. Another drawback with the current implementation of SPC-SBE is the long SPC step resulting from the use of streptavidin and formamide for capture and release of biotinylated fragments, which reduces overall throughput of the technique and restricts its use in an automated setting. In an extension of SPC-SBE, a modification has been made to the SPC step by using monomeric avidin for capture of biotinylated fragments instead of streptavidin (29). This allows release of biotinylated products by a change in pH to substantially reduce time associated with the SPC step. Another problem arises due to uneven crystallization of the 3-hydroxypicolinic acid matrix used for analysis of oligonucleotides. This results in heterogeneity of the sample spot and increases time for analysis owing to the presence of areas where the sample is more concentrated.
In the future, improvements in detection sensitivity of mass spectrometers, coupled with novel techniques for sample preparation and discovery of better matrices for ionization of oligonucleotides, will result in even higher levels of SNP genotyping using SPC-SBE (30)(31). Once large-scale association studies are able to identify SNPs that are important in drug metabolism or as markers for certain diseases, assays for genotyping these SNPs in clinical settings will be needed. The SNPs are likely to be located in different regions of the genome, and efficient approaches for genotyping a limited number of SNPs in large cohorts of individuals will be required. We have demonstrated the applicability of SPC-SBE for use in a candidate gene approach and shown simultaneous genotyping of 50 SNPs that are located over a 9-kb region using a 7-plex PCR. Further innovation, such as the construction of a device to allow faster isolation of extension products, will increase throughput of the procedure. Coupled with automated data collection, the technique will become amenable for use in diagnostic settings.
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Acknowledgments |
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Financial disclosures: None declared.
Acknowledgements: We thank Jingyue Ju and Ming-Cheng Chien (Columbia Genome Center, New York, NY) for generous help with DNA sequencing.
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Footnotes |
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2 Human genes: CYP2C9, cytochrome P450, family 2, subfamily C, polypeptide 9; CYP2A13, cytochrome P450, family 2, subfamily A, polypeptide 13; p53, tumor protein p53 (Li-Fraumeni syndrome; also referred to as TP53).
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