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Molecular Diagnostics and Genetics |
1 Unit of Genomics for Diagnosis of Human Pathologies, Istituto di Ricovero e Cura a Carattere Scientifico Ospedale San Raffaele, Milan, Italy.
2 Dipartimento di Medicina di Laboratorio, Laboratorio di Genetica Medica, Istituti Clinici di Perfezionamento, Milan, Italy.
3 Dipartimento di Medicina Sperimentale e Diagnostica, Sezione di Genetica Medica, Università di Ferrara, Ferrara, Italy.
4 Divisione di Ematologia II, Azienda Ospedaliera Vincenzo Cervello, Palermo, Italy.
5 Laboratorio Genetica Molecolare, Dipartimento di Scienze Biomediche e Biotecnologie, Università degli Studi di Cagliari, Cagliari, Italy.
6 Ospedale Regionale Microcitemico, Cagliari, Italy.
7 Department of Medicine, Center of Translational Medicine, Thomas Jefferson University, Jefferson Medical College, Philadelphia, PA.
8 Diagnostica e Ricerca San Raffaele S.p.A., Milan, Italy.
aAddress correspondence to this author at: Unit of Genomics for the Diagnosis of Human Pathologies, Istituto di Ricovero e Cura a Carattere Scientifico Ospedale San Raffaele, Via Olgettina 60, 20132 Milan, Italy. Fax 39-02-26432640; e-mail ferrari.maurizio{at}hsr.it.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Methods: We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, -87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes.
Results: Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip.
Conclusions: The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing ß-thalassemia in other regions of the world.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We previously showed the feasibility of performing single-nucleotide polymorphism/mutation analysis in a clinical setting with use of a commercially available microelectronic platform, the NanoChipTM Molecular Biology Workstation by Nanogen® (4). This system enables deposition and concentration of charged samples to designated test sites on a 100-microelectrode formatted cartridge (5)(6)(7)(8). Arrays consist of biotin-labeled PCR products amplified from patient DNA and immobilized on test sites through interaction with streptavidin in a permeation layer. Hybridization of specific oligonucleotide probes, complementary to wild-type and mutant sequences, is visualized by fluorogenic indicators.
Our pilot study was instrumental in familiarizing us with this recently developed technology and prompted us to develop a more complex strategy by exploring the potential of this system. By combining multiple addressing and hybridization features, we developed a multiplex fast and highly flexible format that allows identification of nine mutations accounting for >95% of the ß-thalassemia alleles in the Mediterranean area. Our final goal will be the development of a system that covers all or most of the common ß-thalassemia-associated mutations that can be easily applied to the analysis of other common diseases.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Subsequent validation of the assays was performed by analysis of a panel of 250 unrelated individuals, including individuals compound heterozygous or homozygous for any the nine mutations cited above or a wild-type allele. These samples were sent to us by five Italian reference diagnostic laboratories: Dipartimento di Medicina di Laboratorio, Laboratorio di Genetica Medica, ICP (Milan, Italy); Dipartimento di Medicina Sperimentale e Diagnostica, Sezione di Genetica Medica, Università di Ferrara (Ferrara, Italy); Laboratorio Genetica Molecolare, Dipartimento di Scienze Biomediche e Biotecnologie, Università degli Studi di Cagliari (Cagliari, Italy); Ospedale Regionale Microcitemico (Cagliari, Italy); and Divisione di Ematologia II, Azienda Ospedaliera Vincenzo Cervello (Palermo, Italy). The samples had been genotyped previously at these laboratories with other established protocols, such as allele-specific hybridization (dot blot and reverse dot blot), amplification refractory mutation system, restriction enzyme methods for detection of common mutations, or denaturing gradient gel electrophoresis coupled with direct sequencing for rare molecular defects (9)(10)(11)(12)(13). Some of these samples carried 16 additional mutations in the ß-globin gene [hemoglobin (Hb)S, HbC, HbD, Hb Lepore, -101C>T, -87C>T, -29A>G, cd5delCT, cd8-9insG, cd30G>C, IVS1-5G>C, IVS1-130G>C, cd44delC, cd76delC, IVS2-654C>T, or IVS2-848C>A]. We performed the microchip analysis in a blinded fashion because results of previous genotypes were not available to us until the end of the validation procedure.
dna extraction
Genomic DNA was isolated from 10 mL of EDTA-anticoagulated blood by standard phenol–chloroform methods, by a salting-out protocol (14), or by commercial reagents [Blood & Cell Culture DNA Mini Kit (QIAGEN GmbH); Nucleon BACC3 (Amersham Biosciences Europe GmbH)].
pcr conditions
The ß-globin gene regions (promoter, exons 1 and 2, introns 1 and 2) containing the mutations of interest were amplified in four reactions using optimized sets of primers (Table 1 in the Data Supplement available with the online version of this article at http://www.clinchem.org/content/vol50/issue1/), allowing us to analyze all nine mutations of interest. Fig. 1
shows the location of each amplicon. Base composition and primer length were designed to allow uniform thermocycling conditions for all fragments. For each set, one of the primers was 5' biotinylated.
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Polymerase chain amplification was in 50-µL reactions containing 100 ng of DNA, 200 µM deoxynucleotide triphosphates, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 1.3 U of thermostable DNA polymerase (AmpliTaq Gold; Applied Biosystems), and 20 pmoles of each primer. Cycling was as follows: 1 cycle of 95 °C for 10 min; followed by 35 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s; and 1 cycle of 72 °C for 10 min.
pcr multiplexing features
To simplify and automate the analysis, we tested different PCR multiplexing formats. Because of partial overlapping of amplicons 1 and 2 and competition of primer sets between the two adjacent amplicons 1 and 4, PCR multiplexing was possible only by independent processing of two duplexes, one including amplicons 1 and 3 and the other amplicons 2 and 4. PCR cycling conditions were the same as described above. For the multiplex format, 20 pmoles of each primer set were used except for fragment 2, for which 40 pmoles were used.
pcr product purification
After the amplification process, each amplicon was purified and desalted with use of a 96-well plate (Multiscreen-PCR Plates MANU 030) coupled with the Multiscreen Separation System (Millipore Corporation). We then mixed 30 µL of each sample with histidine to a final concentration of 50 mmol/L. For the multiplex format, the two duplexes were pooled together in the same well of the plate. This allowed us to address the mixture of the four amplicons to the same pad as a unique sample. Because of the high number of samples used for assay set up and validation, gel agarose inspection to check for amplification and purification was randomly performed on 
10% of the samples. Sample preparation, including amplification, purification, and electrophoresis on agarose gels, took 3.5 h for 96 samples.
instrumentation and microchip addressing
For microchip analysis, we used the NMW 1000 NanoChipTM Molecular Biology Workstation and NanoChip cartridges fabricated by Nanogen, Inc. (5)(8). This system enables deposition and concentration of charged target samples to designated test sites on a 100-microelectrode formatted cartridge. Details of the instrumentation and protocols for loading and deposition have been described previously (4)(6)(7).
Samples were electronically placed on the chip through a loader and electrophoresed by positive bias direct current to selected pads, where they remained embedded through interaction with streptavidin in the permeation layer. For both optimization and validation of the assays, each sample was loaded and analyzed in duplicate on two different pads, and a heterozygous control was included in each assay. The whole process is automated and takes 3.5 h/chip.
probe design
A total of nine sets of reagents, each including wild-type and mutant reporters and specific stabilizer oligonucleotides (Table 1 in the online Data Supplement), were designed with the help of free programs available on the Internet [DNAmfold server (http://www.bioinfo.rpi.edu/zukerm/rna) and OligoAnalyzer 3.0, by Integrated DNA Technologies (http://www.idtdna.com)] to represent the nine different mutations in the ß-globin gene. Within each pair of wild-type and mutant probes, base length was adjusted to obtain more similar melting behaviors. In the case of the two set of probes detecting a deletion (cd6delA and cd8delAA), the mutant probes were designed to encompass the deleted base(s) and include an additional base to display a melting behavior similar to the wild type. The microchip probe design requires the use of one or two stabilizer oligonucleotides (-87C>G mutation) to open the secondary structure of the template DNA specifically addressed to the DNA region surrounding the mutation of interest. For three mutations, the sequence variation was localized at the 5' (-87C>G) or 3' (IVS2-1G>A, IVS2-745C>G) terminus of the probes because in these cases the basestacking energy allowed a more stable probe-to-template interaction. For the remaining mutations, a dot-blot format was chosen, with the base variation located within the probes.
Classic 5' Cy3 and 5' Cy5 labeling of the wild-type and mutant reporters, respectively, was used for all mutations except for -87C>G, for which 3' 6-carboxytetramethylrhodamine/Bodipy650/665 labeling of the wild-type and mutant reporters, respectively, was used because of secondary structures at the 5' end of the target strand that could interfere with the hybridization process of probes.
hybridization and thermal stringency
After electronic addressing for binding of biotinylated products to streptavidin-containing gel pads, PCR products were denatured in situ by incubation with 0.1 mol/L sodium hydroxide for 3 min. For hybridization, the cartridge was incubated with stabilizers and reporters (1 µmol/L each) in high-salt buffer (50 mmol/L sodium phosphate, pH 7.4, 500 mmol/L sodium chloride) for 3 min at room temperature. To detach all fluorescent probes not perfectly matched to the template DNA, we performed a thermal stringency step specifically for each mutation by appropriately raising of the temperature inside the chip (ranging from 27 to 44 °C; Table 1 in the online Data Supplement). Hybridization was detected by automated fluorescence scanning.
fluorescence detection and interpretation of results
Quantitative analysis of the hybridization results was performed by use of dedicated software accessed through the Internet. Microchips were scanned for all mutations at medium photomultiplier tube gain at an accumulation time of 256 µs for both fluorochromes. Complete processing of the chip (100 pads) took 4 h (software version 1.09.08). With the latest version of the software (1.12.16), the addressing and detection processes have been shortened, and the workstation can analyze four microchips in 1 work day. After controlling for background (addressing buffer with no DNA template) values by subtraction from the signal for each pad, alleles were assigned by evaluating the ratio of specific to nonspecific hybridization. Only samples displaying a signal-to-noise ratio >5 were considered. As a default, samples demonstrating a wild-type:mutant signal ratio between 1:1 and 2:1 were assigned as heterozygotes, whereas samples with a wild-type:mutant signal ratio >5:1 or >1:5 were assigned as wild-type homozygotes or mutant homozygotes, respectively. Samples with a wild-type:mutant signal ratio between 1:2 and 1:5 were not scored (no call). All samples were analyzed in duplicate, after being addressed to two different pads. The mean value from the two pads is automatically calculated by the software for sample genotyping. Hybridization and fluorescence detection take 20 min/chip.
multiple addressing format
The four uniplex PCRs (described in Fig. 1
as 1–4) were sequentially addressed and hybridized to the same microchip pad. Before each addressing, we stripped the previous probes by incubating the chip with 0.1 mol/L sodium hydroxide for 3 min.
multiple hybridization format
In multiple hybridization protocols on the same chip, more than one set of probes were assayed in the singleton PCR format. In this case, because three of four amplicons in our system may contain more than one mutation, only one pair of mutant and wild-type probes can be evaluated per amplicon at each time. We therefore first loaded each of the four amplicons described in Fig. 1
, amplified them singularly on different pads, and hybridized all pads simultaneously with a mixture containing four pairs of wild-type and mutant probes (i.e., cd39, IVS1-6, IVS2-745, and -87), each specific for only one mutation per amplicon. After passive multiple hybridizations with each set of probes, thermal conditions specific for the probe with the lower melting temperature (i.e., 27 °C) were applied, and fluorescence was detected at the corresponding pads. The scanning process was repeated in succession at the increasing temperatures specific for each set of probes (i.e., 31, 35, and 36 °C). After the pad was stripped as described above, we performed a second round of simultaneous hybridizations with a mixture of different pairs of wild-type and mutant probes specific for three additional mutations (IVS1-110, IVS1-1, and cd6). Lastly, hybridization with the two remaining pairs of probes (IVS2-1 and cd8) was also performed.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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13% of those emitted by the shorter ones (data not shown). This could be ascribed to the greater quantity of amplified DNA bound to each test site, which could give rise to complex secondary structures interfering with probe hybridization. In fact, a single amplicon encompassing all nine mutations did not give sufficiently reliable results. The best compromise consisted of two amplicons of 459 and 255 bp, each allowing detection of three mutations; one amplicon (303 bp) allowing detection of two mutations; and one (259 bp) for the remaining mutation, as shown in Fig. 1
. Fluorescent signals corresponding to these fragments were 47% of those emitted by the shortest ones (data not shown). Quantification of the fluorescence emitted from each sample indicated a >50-fold difference in mean intensity between matched and mismatched reporters. Assay conditions were optimized for the correct identification of either mutant or wild-type alleles in 700 known control samples, and distinct fluorescence patterns were obtained for all available genotypes.
blinded validation
In addition to the previously genotyped 700 samples, the biochip assays were blindly validated on 250 individuals randomly chosen from wild-type samples or samples that were mutant homozygous or heterozygous or compound heterozygous for one of the nine mutations listed in Table 1 in the Data Supplement, with complete concordance of results.
In a total of 2250 analyses (250 sample for each of the nine mutations), the ratio for differentiating homozygotes was >20:1 for 99% (2031 of 2056) of the samples. The ratio for heterozygotes was >1:1 and <1:1.5 for 99% (192 of 194) of the samples. The signal-to-noise ratios for all assays were >20:1 for 99.5% (2137 of 2250) of the samples. Fluorescence signals for the remaining samples were above the thresholds (see Patients and Methods), allowing unambiguous genotyping in all cases. An overall 1% no-call rate was obtained because of either a lack of or insufficient amplification or sample loss during purification, as revealed by subsequent agarose gel electrophoresis. Reamplification of the same samples gave the appropriate results. The overall repeat rate was 1%.
No cross-hybridization for any of the nine sets of probes (wild-type and mutant) was obtained, even in the case of patients carrying the -87C>T, HbS (cd6A>T), HbC (cd6G>A), cd8-9insG, and IVS1-5G>T mutations, which involve the same or adjacent nucleotides with respect to the nine mutations tested in the present study (-87C>G, cd6delA, cd8delAA, and IVS1-6T>C).
amplification, addressing, and hybridization multiplexing features
Different multiplexing formats were explored. Duplexed amplification of the four regions of the ß-globin gene (described in Fig. 1
as 1–4) and their pooling before purification allowed us to address each single pad with the multiplex PCR products, which were then sequentially hybridized with each set of wild-type and mutant probes. An example of results obtained for the detection of the cd39, IVS1-110, IVS1-1, and IVS1-6 mutations in the same five samples is shown in Fig. 2
. This allowed interrogation of one DNA sample per pad for all nine mutations, giving complete analysis of the nine mutations in 100 samples on one chip.
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We also checked the possibility of using the same chip with different samples more than once. The four uniplexes (1)(2)(3)(4) could be sequentially addressed and hybridized to the same pad, after stripping of the previous probes. Multiple addressing takes the same time as single addressing but allows reuse of the same pad, and thus the same chip, up to four times. An example of multiple addressing is shown in Fig. 3
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For both formats, absolute fluorescence values and signal-to-noise ratios (>20:1) were the same as those already reported for single analyses of one amplicon per pad.
We also simultaneously hybridized the same chip with several sets of wild-type and mutant probes. By this approach, three rounds of multiple hybridizations were performed, the first with four probe sets, the second with three, and the last with the two remaining probe sets. Multiple hybridization uses the same number of pads as the single format but allows shortening of the analytical procedure by reducing from nine to three the number of hybridization steps. With this approach, each sample gave absolute fluorescence signals slightly lower (20% medium range) than those obtained with the singleton hybridization approach. This may be ascribed to the use of a mixture of probes, which all nonspecifically bind the target DNA during passive hybridization at room temperature. This probably lowered the number of target molecules available for specific hybridization. By raising the temperature in subsequent steps and washing with a low-salt buffer, only the specific set of probes was bound to the target, as demonstrated by the absence of any cross-hybridization signal in all of our assays. All values were above the threshold, allowing correct identification of all nine mutations.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The discriminative properties of all microchip probes were tested extensively, and the method described here provided unambiguous detection of complex heterozygous mutation combinations with no cross-reactivity, even in the case of different substitutions affecting the same positions as or nucleotide positions adjacent to the ones interrogated by our probes.
Moreover, with respect to large-scale screening applications, we successfully developed several alternative multiplexing features, allowing us to reduce time and costs.
We first established conditions for simultaneous amplification of different fragments in the same tube. Two duplex PCR reactions were pooled, purified, and addressed to the same pad, allowing analysis of all nine mutations in a unique sample on the same test site, thus reducing addressing time to one-fourth. In addition, we had success when we readdressed the same pad several times with different amplicons or, alternatively, performed multiple simultaneous hybridizations on the same chip with up to four sets of probes. The combination of all such properties may allow the user to maximize the number of mutations typed per sample and/or to increase the number of addressable test sites, according to the necessity.
The developed platform provides a completely automated device that allows the scanning of up to four chips (400 samples) in a single day, thus facilitating the screening of a large number of samples in a highly frequent disease such as ß-thalassemia. Additionally, the ability to control individual tests permits customization of each chip according to daily necessity, providing an open flexible platform that may be individually designed according to each local situation. The nine mutations analyzed in the present study would identify 95% of individuals with ß-thalassemia in the European Mediterranean countries. Most of the DNA regions of the ß-globin gene impacted by ß-thalassemia mutations are already included in the amplicons designed for the present work. This implies that additional mutations can be analyzed easily in the multiplex format by simply adding the specific probes, thus allowing expansion of the panel of the mutations to be identified. Of note, the protocol we present here can be quickly transferred and adapted to other diagnostic laboratories.
In summary, the present work represents an important step toward the development of a diagnostic ß-thalassemia chip based on the principle of hybridization of probes to DNA molecules electronically immobilized on microchips that target numerous mutations.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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-thalassemia repository (9th ed., part I). Hemoglobin 1998;22:169-195.[ISI][Medline]
[Order article via Infotrieve]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and mutant (
) probes for each mutation are shown. Genotypes were as follows: sample 1, cd39/IVS1-1; sample 2, IVS1-110/IVS1-1; sample 3, cd39/cd39; sample 4, cd39/IVS1-6; sample 5, IVS1-110/IVS1-110. (Bottom), black (wild type) and gray (mutant) signals, the number of pads used for the analysis, the black and gray ratios (B:G), and final genotyping results for IVS1-110 are listed for each sample. Sample 2 was used as internal heterozygous control for normalization of wild-type and mutant signals for IVS1-110G>A.