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Molecular Diagnostics and Genetics |
1 Genomic Unit for the Diagnosis of Human Pathologies, San Raffaele Scientific Institute, Milano, Italy; 2 Istituto di Chimica del Riconoscimento Molecolare (ICRM), C.N.R., Milano, Italy; 3 Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA; 4 Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA; 5 Dipartimento di Medicina Sperimentale e Patologia, Universita’ degli Studi "La Sapienza," Roma, Italy; 6 Ricerca e Diagnostica San Raffaele, Milano, Italy; 7 Universita’ Vita-Salute San Raffaele, Milano, Italy.
aAddress correspondence to this author at: Genomic Unit for the Diagnosis of Human Pathologies, DIBIT2, San Raffaele Scientific Institute, Via Olgettina 60 Milan, 20132 Milan, Italy. Fax 0039 02 26434351; e-mail cremonesi.laura{at}hsr.it.
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Abstract |
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Methods: We developed an array-based genotyping assay based on glass slides coated with copolymer (N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate). The assay consists of an LDR with genomic DNA followed by a universal PCR (U-PCR) of genomic DNA–templated LDR product. The LDR occurs in the presence of 3 primers for each sequence variant under investigation: 2 distinguishing primers (allele specific and perfectly complementary to wild-type and mutant alleles) and 1 common locus-specific primer. The 2 allele-specific primers have different capture sequences for binding different complementary probes on a tag array. The LDR product templated from genomic DNA is made fluorescent during the U-PCR via incorporation of a Cy5-labeled universal primer into all LDR products; detection occurs on the coated glass slides.
Results: The assay was designed to detect 7 prevalent mutations in the β-globin gene (HBB, hemoglobin, beta) in a multiplex format, and signals for the different alleles are detected by their fluorescence. The assay was applied to 40 genomic DNA samples from both control individuals and patients with known β-thalassemia mutations. Results show good correspondence between the patients’ genotypes as assessed by DNA sequence analysis and those generated from the LDR assays.
Conclusions: The developed technology allows accurate identification of sequence variants in a simple, cost-effective way and offers good flexibility for scaling to other applications with different numbers of single-nucleotide polymorphisms or mutations to be detected.
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Introduction |
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In particular, a large variety of methods have been developed to detect mutations in the human β-globin gene (HBB, hemoglobin, beta) that cause thalassemia, a major health problem worldwide. Traditional enzymatic PCR-based methods include an allele-specific amplification refractory mutation system(2), restriction endonuclease analysis of PCR-amplified products(3), and denaturing gradient gel electrophoresis(4). Automated sequencing represents the gold standard but is still too expensive for routine application. Pyrosequencing(5) is cheaper and easily amenable to automation(6). Several microarray systems that have been developed for the detection of human globin gene mutations are appropriate for medium- or high-throughput applications, and the systems that are commercially available include a microelectronic array system, an arrayed primer extension–based system, and systems involving tagged single-base extension with hybridization to either universal glass or flow-through microarrays(7).
Assays that do not require expensive hardware and proprietary reagents but still provide high specificity and sensitivity are needed for smaller-scale analyses. The possibility of multiplexing the genotyping assays may allow further cost reductions and increases in throughput. Locus-specific PCR is still a rate-limiting step, especially when it is coupled with the development of multiplexing assay formats. This problem may be overcome by allele-specific oligonucleotide ligation.
We describe a simple technology that addresses the need for cost-effectiveness by offering good flexibility and scaling to different numbers of single-nucleotide polymorphisms or mutations to be detected. This assay detects the βS allele and 6 prevalent mutations that cause β-thalassemia(8), one of the most common genetic diseases in the Mediterranean basin. A multiplex ligation detection reaction (LDR)1 strategy has been adopted to detect these mutations simultaneously in a single tube.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Because of the high fidelity of the ligase at the nick junction, a perfectly matched genomic DNA–templated base pair will be ligated to the common primer, whereas a mismatched template/primer base pair will not. LDR achieves linear amplification through repeated steps of genomic-DNA template denaturation, primer annealing, and ligation(11). An amplification step is then needed to increase the yield of the LDR product so that it can be analyzed on the array. We performed a universal PCR (U-PCR) with incorporation of a fluorophore (Cy5) coupled to the universal primer. In addition, the U-PCR was done asymmetrically to favor amplification of the strand to be captured on the array.
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Common glass microscope slides were activated with a polymeric coating produced by physical adsorption of a N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS), and 3-(trimethoxysilyl)propyl methacrylate (MAPS) copolymer, which provides an active surface on which amino-modified oligonucleotides can be covalently immobilized(12)(13)(14)(15). Slides were then spotted with selected probes. After incubation of the LDR/U-PCR product on the coated slides, the fluorescence signal was measured by scanning. Ligation reactions were performed first in a uniplex format with 10 DNA samples. About 100 different LDR experiments were necessary to optimize the conditions for detecting the 7 mutations. The protocol was then converted into a multiplex format for the simultaneous detection of all 7 mutant alleles.
genomic dna samples
After we obtained informed consent according to the local Institutional Review Board Committee, we collected genomic DNA samples from control individuals with a wild-type HBB gene and from patients homozygous or heterozygous for a variety of HBB mutations (OMIM accession no. 141900): IVS-I nt 1 (G>A), IVS-I nt 6 (T>C), IVS-I nt 110 (G>A), codon 39 (C>T), IVS-II nt 1 (G>A), IVS-II nt 745 (C>G), and Hb S (A>T). The alternative nomenclature for these mutations according to the Human Genome Variation Society (http://www.hgvs.org) is as follows: c.92 + 1G>A, c.92 + 6T>C, c.93–21G>A, c.118C>T, c.315 + 1G>A, c.316–106C>G, and c.20A>T, respectively.
Samples had previously been genotyped by standard cycle-sequencing methods. We used 10 DNA samples to develop LDR and U-PCR conditions and then retrospectively analyzed 40 DNA samples with this method. Before ligation, we fragmented each sample by heating for 5 min at 99 °C.
ligase detection reaction
The LDR is performed in a 10-µL reaction containing 200 ng of fragmented genomic DNA, 1 nmol/L of each distinguishing primer (wild-type and mutant), 2 nmol/L common primer, 1x Pfu ligase buffer [20 mmol/L Tris-HCl, pH 7.5, 20 mmol/L KCl, 10 mmol/L MgCl2, 1 mL/L Igepal, 10 µmol/L ribose ATP, and 1 mmol/L dithiothreitol (Strategene Cloning Systems)], 40 g/L polyethylene glycol 8000, and 2 U Pfu ligase (Stratagene Cloning Systems). The reaction was heated to 95 °C for 2 min and then subjected to 20 cycles of 95 °C for 15 s followed by 4 subcycles of 60 °C for 30 s and 70 °C for 10 s(16). Pfu ligase was inactivated at 99 °C for 10 min. We treated LDR products with exonucleases to remove unligated LDR primers before the PCR by adding 5 U 
exonuclease and 5 U exonuclease I to each sample in 1x exonuclease buffer (67 mmol/L glycine-KOH, pH 9.4, 2.5 mmol/L MgCl2, and 0.1 mL/L Triton X-100) in a 20-µL final volume, incubating at 37 °C for 2 h, and then incubating for another 30 min at 80 °C to inactivate the enzymes. The final MgCl2 concentration during the exonuclease step was 7.5 mmol/L because of the contributions of the LDR and exonuclease buffers.
ldr primers
Phosphothiolated blocked ends were engineered at the 5' ends on the allele-specific primers (wild-type and mutant) and at the 3' end on the common locus-specific primer (see Tables 1 and 2 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol54/issue10). In addition, at the ends of the LDR primers we inserted U-PCR priming sites, one complementary to the A sequence at the 5' end on all allele-specific primers and the other complementary to the B sequence at the 3' end of each common locus-specific primer (see Fig. 1A
). Each allele-specific primer (wild-type and mutant) also has a different "zip-code"(17) for binding to a complementary capture probe on a universal tag array (Fig. 1
).
universal pcr
We added 10 µL of the LDR/exonuclease product to the PCR mix in a 50-µL final reaction volume containing 1x AmpliTaq Gold Buffer II (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl; Applied Biosystems), 200 µmol/L of each deoxynucleoside triphosphate, 360 nmol/L universal A primer (5'-CGACTCTAATACGACTCACTATAGGG-3'), 36 nmol/L universal B primer (5'-GGGTTGTACTACATTCGTGCGATGG-3'), 2 mmol/L MgCl2, and 2.5 U AmpliTaq Gold (Applied Biosystems). The Cy5 fluorophore was coupled to the A universal primer. The cycling program was 95 °C for 10 min and 35 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. We checked the PCRs by electrophoresing 8-µL aliquots on agarose gels (20 g/L) stained with ethidium bromide. The PCR product (about 42 µL) was precipitated with ethanol and resuspended in 10 µL H2O for hybridization to the array.
coating of glass slides
Copolymer (DMA-NAS-MAPS) was synthesized and characterized as previously described(12). The coating of glass slides requires 2 steps: (a) surface cleaning and pretreatment and (b) adsorption of the polymer. Glass microscope slides (Sigma–Aldrich) were cleaned by immersion in pure ethanol for 30 min, treated with 1 mol/L NaOH for 30 min and 1 mol/L HCl for 1 h, washed with water, and dried. The pretreated glass slides were then immersed for 30 min in 10 g/L copolymer solution (DMA-NAS-MAPS) in an aqueous solution of 20%-saturated ammonium sulfate. Finally, slides were washed extensively with water and then dried for 20 min under vacuum at 80 °C.
microarray preparation
We selected 14 different oligonucleotides from the GeneFlexTM Tag Array sequence collection (Affymetrix) as capture probes to be spotted on the glass slides (see Table 3 in the online Data Supplement). The capture probes, which had been amino-modified at the 3' end (MWG-Biotech), were dissolved in 1x printing buffer (150 mmol/L sodium phosphate, pH 8.5) and printed in 5 replicates with a piezoelectric spotter (SciFLEXARRAYER S5; Scienion). Spotting was done at 20 °C in an atmosphere of 50% humidity. The zip-codes were coupled to the arrays by incubating overnight in a hypersaturated NaCl chamber at room temperature. After incubation, all residual reactive groups of the polymer coating were blocked by dipping the slides in prewarmed blocking solution (50 mmol/L ethanolamine, 0.1 mol/L Tris, pH 9.0) at 50 °C for 15 min and then rinsed twice in distilled H2O. Slides were washed in prewarmed postcoupling washing solution [4x saline sodium citrate (SSC) solution (0.6 mol/L NaCl and 0.06 mol/L sodium citrate), 1 g/L SDS] at 50 °C for 15 min, rinsed with distilled H2O, dried by centrifugation at 100g for 3 min, and stored in a desiccator.
hybridization
The resuspended LDR/PCR product (10 µL) was added to a hybridization mix in a final reaction volume of 15 µL containing 2x SSC buffer, 1 g/L SDS, and 0.2 g/L BSA. The mix was heat-denatured at 99 °C for 10 min and snap chilled on ice. Afterward, the prepared arrays were coated with the hybridization solution described above in a humid chamber for 1 h at 20 °C. Arrays were removed from the hybridization chambers and soaked briefly in 4x SSC buffer to remove the lifter strips, washed twice for 5 min in 2x SSC buffer containing 1 g/L SDS solution, dipped briefly in 0.2x SSC and then in 0.1x SSC, and centrifuged at 100g for 3 min.
image scanning and data analysis
We used the red laser of the ScanArray Lite (PerkinElmer) to scan the hybridized slides for the Cy5 dye (633 nm excitation, 670 nm emission). Slide images were acquired at 65% laser power and 60% photomultiplier tube gain, and 16-bit TIFF images were analyzed at 10-µm resolution. Fluorescence intensities were extracted with the scanner software. Data analysis was performed as follows: The local background was subtracted from the fluorescence intensity of each spot, and only values displaying a signal-to-noise ratio of >3 were considered. The signal-to-noise ratio was the ratio of the spot intensity to the SD of the local background of all spots on the microarray. The fluorescence intensities of 5 spots were then averaged, and the SD was calculated.
signal normalization
To balance the different efficiencies of the LDR primers and capture probes, we first established normalization factors to be applied to the analysis of data for unknown DNA samples. To assess interslide variation, we hybridized 7 known DNA samples, each heterozygous for one mutation, in triplicate on the same day and on 3 different slides. After subtracting the background signal, we measured the mean fluorescence intensity as previously described and then calculated the ratio (r) of the wild-type fluorescence (W) to the mutant fluorescence (M) for each analysis (e.g., r1 = W1/M1). The mean of the 3 replicate ratios per heterozygote was estimated as the normalization factor (nf) [i.e., nf = (r1 + r2 + r3)/3].
genotype assignment
After setting the nf for each mutation, we analyzed the 40 DNA samples according to the protocol. We averaged the fluorescence intensities of the 5 spots (Wa and Ma) and calculated the normalized mutant fluorescence (Mn) (i.e., Mn = Ma x nf). Ratios of the wild-type signal to the normalized mutant signal were then estimated for each sample (Wa/Mn). Samples displaying a Wa/Mn signal ratio between 1:1 and 1:2 were assigned as heterozygotes, whereas those with a Wa/Mn ratio of 5:1 or 1:5 were assigned as wild-type or mutant homozygotes, respectively. Samples with a Wa/Mn signal ratio between 1:2 and 1:5 were not scored (no call)(18).
safety considerations
Sample preparation and assay protocols did not involve any hazardous procedures or use of toxic reagents. The monomers used for DMA-NAS-MAPS copolymer preparation are toxic and must be handled according to the manufacturers’ instructions.
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Results |
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. All 40 DNA samples produced fluorescence signals that could be scanned and analyzed by the software. Normalization was applied to each sample for every mutation, and the correct genotype was assigned to all samples for all alleles, with the exception of 5 samples, in which 1 of the 7 genotypes was ambiguous because the ratio of the wild-type signal to the mutant signal was between 1:2 and 1:5 and therefore scored as a no call. We observed no cross-hybridization for any of the 7 sets of probes (wild-type and mutant) and obtained no false-negative or false-positive results.
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Fig. 2
presents an example in which 7 different sites were interrogated simultaneously in a single patient sample (i.e., IVS-I nt 6, IVS-II nt 1, codon 39, IVS-I nt 1, IVS-I nt 110, codon 6 (hemoglobin S), and IVS-II nt 745). The patient is a compound heterozygote for mutations (M) at IVS-I nt 110 and IVS-II nt 745 and should yield the expected pattern outlined in Fig. 2A
. Each row has 5 replicate spots for each site being interrogated. Results of the fluorescence scan of the slide in Fig. 2B
shows the expected pattern depicted in Fig. 2A
. Five of the interrogated sites show only the wild-type alleles while both wild-type (N) and mutant (M) alleles are seen at positions IVS-I nt 110 and IVS-II nt 745, as expected for a compound heterozygote at these 2 sites. The normalized results shown in Fig. 2C
clearly show the distinction in the interrogated sample between N/N and N/M genotypes at the different HBB sites tested.
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Discussion |
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As an example of a genetic application, we evaluated the simultaneous detection of 7 prevalent HBB gene mutations. The overall strategy is made possible by functionalizing the LDR primers with multiple sequence-specific elements. Each allele-specific LDR primer has a unique zip-code–capture sequence that directs the successfully ligated and amplified product to a particular site on the array to bind the complementary probe spotted on the slide(17). These zip-code–capture sequences are all 20-mers that have similar melting temperature values and have sequences that avoid any cross-hybridization. In addition, we inserted 2 different U-PCR priming sites at the ends of all LDR primers (A, at the 5' end of all allele-specific primers, and B, at the 3' end of all common locus-specific primers; see Fig. 1A
) to facilitate U-PCR amplification of only correctly ligated molecules. We also built phosphothiolated blocked ends into each LDR primer, at the 5' end on the allele-specific primers and at the 3' end on the locus-specific common primers, to protect correctly ligated doubly blocked LDR products from exonuclease digestion. This strategy facilitates exonuclease removal of unligated LDR primers before the U-PCR of the LDR product. In this way, the unligated primers are removed because only one end is blocked on each primer.
We subsequently carried out 40 experiments by performing the sample preparation, hybridization, data analysis, and normalization steps as described above. Each sample was interrogated for the 7 wild-type and 7 mutant alleles for a total of 40 x 7 (280) genotypes, leading to the identification of 560 alleles (Table 1
). In this application for the HBB gene, all genotypes were correctly assigned except for 5 samples, in which 1 of the 7 genotypes was ambiguous and scored as a no call. In these cases, the assay detected a variant at the correct site, but it could not assign heterozygosity or homozygosity because the ratio of the wild-type signal to the mutant signal was between 1:2 and 1:5. Interestingly, the overall 1.8% no-call rate in 5 of the 280 analyses involved the detection of the codon 39 mutation, either in homozygotes or heterozygotes. The same 5 samples were correctly genotyped for all of the other wild-type and mutant alleles, thus excluding any problem related to DNA quality in these samples. In any case, we never had a false-positive result, because the 5 no-call samples always involved the codon 39 sequence variation. Analysis of the codon 39 site with this assay may need to be optimized in a uniplex format for both the LDR and hybridization steps. In the present work, we developed a multiplex version of the test, which allowed the detection of 7 variants in a single tube with one hybridization temperature per sample. This result demonstrates the potential applicability of the system for diagnostic purposes.
In conclusion, multiplex LDR was implemented in the present work for the simultaneous detection of the βS allele and 6 β-thalassemia mutations. The assay could easily be scaled up without changing the U-PCR conditions, because all LDR products contain sequences complementary to the U-PCR primers. Additionally, this assay requires only a small amount of DNA and needs only one universal primer coupled to a fluorophore in order to report all genotypes. Moreover, glass slides are very cheap, and hybridization occurs in a short time, even at room temperature.
Our results indicate that this strategy has sufficiently high sensitivity and specificity to differentiate multiple targets simultaneously. The platform also has the potential for direct identification of mutations in unamplified genomic DNA, thus avoiding the U-PCR step.
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Acknowledgments |
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Authors’ Disclosures of Potential Conflicts of Interest: Upon submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: L. Cremonesi, European Commission for the Special Non-invasive Advances in Fetal and Neonatal Evaluation (SAFE) Network of Excellence (LSHB-CT-2004-503243), Telethon (GGP04016); P. Fortina, Kimmel Cancer Center, Pennsylvania Department of Health (SAP4100026302); S. Surrey, NIH (HL-69256), Cardeza Foundation for Hematological Research, Philadelphia, PA.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
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References |
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