High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes
High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genesAnthony P. Shuber+, Lesley A. Michalowsky, G. Scott Nass, Joel Skoletsky, Lisa M. Hire, Steve K. Kotsopoulos, Michael F. Phipps, Dana M. Barberio and Katherine W. Klinger*
Department of Technology Development, Genzyme Genetics, Framingham, MA 01701-9322, USA
Received September 9, 1996;Revised and Accepted November 29, 1996
As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (>500) simultaneously for a large number of known mutations (>100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 [beta]-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in >500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.
Over the last several years, there has been a significant increase in the number of identified, cloned, and characterized genes responsible for inherited diseases in humans. As the number of disease-associated sequences has increased, the number of mutations identified within the genes has likewise increased. In some genes, only one or a few mutations are specifically responsible for the disease phenotype (e.g. sickle cell anemia ) (1 ). However, in most disease genes, many different causative mutations exist with no single mutation present at a significant frequency within an affected patient population. Therefore, for both research and clinical diagnostic applications, improved methods of mutation analysis are required not only to confirm that a candidate gene truly represents the disease gene, but also to build mutation databases and provide clinical diagnostic assays. In addition, with the increasing number of cancer genes being discovered (2 -4 ), efficient, cost-effective, and highly informative mutation analysis procedures are necessary in order to better understand predisposition and polygenic diseases.
This has led to the development of two broad categories of mutation detection technologies (5 ,6 ). The first group, designed to scan for mutations within a gene, includes single-strand conformational polymorphism (SSCP) (7 ), denaturing gradient gel electrophoresis (DGGE) (8 ), heteroduplex analysis (HET) (9 ), chemical cleavage analysis (CCM) (10 ), ribonuclease cleavage (RNase) (11 ) and direct sequencing of the target (12 ). Although these procedures are highly informative, they can be tedious and are incompatible with high throughput and low cost. Given the need in the clinical diagnostic laboratory to be able to analyze large numbers of samples (>500 samples/analysis) cost-effectively, these scanning procedures are not used currently as routine methods of mutation detection (5 ). In the second group, more direct methods of mutation analysis have been developed such as allele-specific amplification (ASA) (13 ), oligonucleotide ligation assay (OLA) (14 ), primer extension (15 ), artificial introduction of restriction sites (AIRS) (16 ), allele-specific oligonucleotide (ASO) hybridization (17 ) and variations of these procedures. Together with robotics, these methods for direct mutation analysis have helped in reducing cost and increasing throughput when only a limited number of mutations need to be analyzed. However, given that many of the mutations identified in disease genes are rare, for most populations undergoing testing, large numbers of mutations must be analyzed in order to achieve significant detection frequencies.
Unfortunately, such comprehensive multiplex mutation analysis (>100 mutations) cannot be performed by any currently available diagnostic method while retaining the sample throughput and cost-effectiveness needed in a clinical diagnostic laboratory. A potential solution to this problem involves a miniaturized version of the reverse dot-blot procedure (18 ). The `chip' technology, or sequencing by hybridization (SBH), involves the hybridization of a single labeled target to a dense panel of oligonucleotides arrayed on a solid support (19 -23 ). Although this approach is an extremely appealing solution for large multiplex mutation analysis, a number of technical issues must be addressed before it can be applied routinely to clinical diagnostics (24 ). In addition, as with the reverse dot-blot procedure, each sample analysis is performed independently. Therefore, a significant effort is needed in order to develop a procedure that will allow large numbers of samples (100-500) to be analyzed in a single assay.
While our previous modified ASO approach allowed the simultaneous analysis of large numbers of patient samples for multiple cystic fibrosis (CF) mutations (25 ), relatively inefficient independent hybridizations were required to identify specific mutations. Herein, we present a methodology which combines high sample throughput and direct detection of a large number of sequence variants. The multiplex allele-specific diagnostic assay (MASDA) has the capacity to analyze large numbers of samples (>500) for a large number of mutations (>100) cost-effectively in a single assay. Like the more familiar `chip' technologies (19 -22 ), MASDA uses oligonucleotide hybridization to interrogate DNA sequences. However, in contrast to many oligonucleotide array approaches, in the MASDA technology, the target DNA is immobilized to the solid support, and interrogated in a combinatorial fashion with a pool of ASOs (i.e. a single solution of hundreds of different oligonucleotides with each oligonucleotide sequence specific for one mutation). By retaining the forward dot-blot format, we can analyze large numbers of samples (>500) for a large number of mutations (>100) simultaneously. A schematic diagram of the MASDA technology is shown in Figure 1 . During the hybridization, the ASO(s) corresponding to a specific mutation(s) present in a given sample is hybrid-selected from the pool of probes by the target DNA. Following removal of unhybridized ASOs, sequence-specific band patterns associated with the bound ASOs are generated by chemical or enzymatic sequencing, and the mutation or mutations present in the sample are easily identified. Using the gene targets CFTR (26 ), [beta]-globin (1 ), HEXA (27 ), GCR (28 ), ASPA (29 ), BRCA1 (3 ) and FACC (30 ) as a model system, we demonstrate that MASDA not only allows different patient samples with different disease indications to be analyzed in a single assay, but allows the identification of multiple mutations in a single gene or multiple genes in a single patient's DNA sample.
Seven different gene targets, representing eight different diseases, were chosen as a model system for complex mutation detection (Table 1 ). A total of 106 different mutations were analyzed in a single hybridization and detection procedure, referred to as MASDA 106. Although large numbers of mutations have been identified within the majority of disease genes listed, for the purposes of this study a selected number of these mutations were used (Table 1 columns 3 and 4). The specific mutations chosen within each disease gene represented the most clinically relevant for diagnostic applications (except for CF, since the most common mutation [Delta]508 was not included in this assay, and except for BRCA1 where the development of mutation databases with genotype-phenotype correlations are ongoing). The largest number of mutations analyzed resided within the CFTR gene. In addition to the most frequently detected mutations within a CF patient population (Cystic Fibrosis Genetic Analysis Consortium, 1994; unpublished data), additional point mutations were included that lead to premature translation termination, and subsequently a truncated protein product. A total of 33 different amplification products were needed in order to interrogate for the presence or absence of the 106 different mutations (Table 1 column 5). It is important to note, however, that amplifications were performed in a disease-specific manner only, for example, if a patient was suspected to be a CF carrier, the DNA sample was amplified for the CF gene only.
The specific mutations examined within each disease gene are shown in Table 2 . These tables also include the size (bp) of regions amplified, and the primers used for each amplification.
Where applicable, multiplex PCR was performed to reduce the number of PCR reactions needed (Table 1 , column 5). A total of nine reactions facilitated amplification of 33 different loci. All single or multiplex PCR reactions were performed in a disease-specific manner. In other words, individual DNA samples were amplified for the relevant disease gene only, and not for all loci examined in the assay. Examples of the various disease-specific amplification products are shown in Figure 2 . In order to include 66 CF mutations within our study, 17 different regions within the CFTR gene were amplified using two multiplex PCR reactions (Fig. 2 , lanes 1 and 2). Amplicon sizes ranged from 130 to 510 bp. A single amplification product of 1600 bp was sufficient to include 14 [beta]-thalassemia- and two sickle cell anemia-associated mutations within the [beta]-globin gene (Fig. 2 , lane 3). For Tay-Sachs assays, a 2-plex amplification reaction was designed to examine three mutations (Fig. 2 , lane 4). To examine Canavan-associated mutations 3-plex amplification reactions were performed (Fig. 2 , lane 6). A separate 4-plex amplification was performed for five breast cancer susceptibility-related mutations (Fig. 2 , lane 7) and a single 3-plex amplification for Fanconi anemia-associated mutations (Fig. 2 , lane 8). For Gaucher disease, the GCR pseudogene necessitated a 3-plex amplification and an independent, single amplicon amplification. For convenience of analysis, aliquots from both reactions were pooled and electrophoresed in the same lane of the analytical gel (Fig. 2 , lane 5).
The specific mutation present in a positive sample was identified by eluting the hybridized oligonucleotide from each individual dot and directly interrogating the oligonucleotide sequence. In one scheme, the eluted oligonucleotides were attached to a solid support, G and C base-specific chemical modification reactions were performed and the reaction products separated by polyacrylamide gel electrophoresis (32 ). Figure 4 represents an example of the CG-limited sequencing fingerprints produced from some of the oligonucleotides eluted from the mutation-specific positive control samples in Figure 3 (CF30, CF31, TS1, TS2, TS3, BT2, BT3*, BT6 and BT7). As shown in Figure 4 , the oligonucleotide eluted from each dot generated a characteristic fingerprint which unambiguously identified the specific mutation present in the positive sample DNA. Unique band patterns were generated for each of 106 ASOs (data not shown). Sequence analysis of all 106 eluted oligonucleotides verified that a positive result from the single pooled hybridization represented specific oligonucleotide hybridization with no significant cross-hybridization between different probes. In addition, one sample containing two [beta]-globin mutations (Fig. 4 , lane BT3*) generated a unique fingerprint made up of two superimposed oligonucleotide-specific band patterns. This demonstrated that a compound heterozygote genotype was readily identified using this technique.
To validate the procedure, we performed a blinded analysis to assess the ability of the MASDA technique to identify mutations as envisaged in the diagnostic setting. More than 500 samples whose genotype had been established previously using other techniques were obtained from clinical collaborators. The genotypes remained unknown to the laboratory personnel. After the analysis, the MASDA results were compared with the known genotype. Figure 6 represents the hybridization results generated from analyzing >500 different DNA samples for the presence of 106 different mutations, in a single hybridization assay. All samples known to carry one of the 106 different mutations were identified correctly as positive in the hybridization (Fig. 6 ). The specific mutations were identified correctly in each positive sample by performing the chemical modification and cleavage procedure (data not shown). It is significant to note that no increase in non-specific background was observed when sample throughput was increased to >500 samples in a single hybridization assay.
We believe that the MASDA approach provides a major breakthrough in mutation detection. The combination of the forward dot-blot, complex simultaneous probe hybridization and direct mutation detection for the first time solves the dual problems of parallel, multiple sample analysis and parallel multiplex mutation detection. In contrast, a significant limitation in almost all of the currently available techniques is the inability to analyze a large number of samples simultaneously for a large number of mutations. The traditional dot-blot, wherein the PCR products are bound to a filter membrane and hybridized with allele-specific probes, is a very effective format for the analysis of large numbers of samples. However, in a standard forward dot-blot procedure, a separate hybridization is performed for each allele or mutation of interest. Because the number of probes required for genetic diagnosis is large, this procedure is very cumbersome. Previously we described a methodology for CF testing which pooled together multiple mutation-specific oligonucleotides into a single hybridization (25 ). This format retains the capacity for large sample throughput while reducing the number of hybridizations involved in performing multiple mutation analysis. Even with this approach, the limited number of reporters available to discriminate between probes necessitated secondary independent hybridizations on all positive samples to identify the specific mutation present, reducing the cost-effectiveness of the methodology. The ability of the reverse dot-blot and the miniaturization of reverse dot-blots (oligonucleotide arrays) to analyze multiple probes is an appealing solution to this problem. However, in this approach, a separate hybridization is performed for each sample, thus eliminating the capacity for large sample throughput.
In the MASDA approach, the disadvantages of both individual sample hybridizations and independent probe hybridizations are avoided. By eluting and interrogating the sequence of the mutation-specific oligonucleotides hybridized to a patient's DNA sample, MASDA eliminates the need for secondary independent hybridizations. Therefore, in a single day, hundreds of different samples can be analyzed simultaneously in a single hybridization containing a complex mixture of hundreds of mutation-specific oligonucleotides. Also, by generating short and unique band patterns for the hybridized and eluted oligonucleotides, multiple samples can be analyzed by stagger loading samples across multiple lanes of a gel. Therefore, using currently available automated sequencers, specific mutation identification can be performed easily on hundreds of positive samples at a rate in the range of 150-900 samples/h.
When designing DNA diagnostic assays for widespread use in clinical laboratories, several parameters need to be considered in addition to high throughput sample capacity and multiplex mutation detection. These include ease of performance, overall assay economies of scale and ease and cost of modification [both in the assay design process and in implementation of the modification(s) in the clinical lab]. This is very important in a field such as genetic diagnostics, where both the number of relevant genes and mutations identified in each gene change rapidly, as do available instrumentation and detection systems. With these considerations in mind, MASDA has been designed to be a flexible, modular platform, allowing all of these challenges to be addressed.
There is minimal development necessary for the addition of new allele-specific oligonucleotides to a probe pool, and no incremental labor cost to perform the improved/expanded test. For example, optimization of the oligonucleotides in MASDA 106 involved nothing more than an independent hybridization of each oligonucleotide to determine the concentration necessary to yield a comparable signal to other mutation-specific probes in the pool. This allows new mutations easily to be added to any diagnostic assay. Because the hybridizations are performed with the oligonucleotide probes in solution, it is possible to mix and match probes on demand, therefore allowing clinical laboratories to customize diagnostic assays cost-effectively. Thus the assay can be readily modified without incurring the significant costs associated with the design and manufacture of new hardware or disposables. There is also flexibility in sample preparation and target detection. The sample nucleic acid can be either DNA or RNA. For the purpose of performing multiplex target amplifications, PCR was utilized as the amplification procedure for this work. However, MASDA is compatible with any target amplification technology, and does not require any processing of amplification products prior to mutation detection and identification. This becomes a very important issue when large numbers of samples need to be analyzed in a single assay. Since the sample nucleic acid does not need to be fragmented, long PCR products can be analyzed, as well as the multiplex amplicons demonstrated in this study.
A variety of different reporter groups and detection methods are compatible with MASDA. For example, while the data presented herein used an isotopic format, MASDA is equally powerful with non-isotopic detection methods such as chemiluminescence and fluorescence, and appropriate instrumentation for sequence analysis (data not shown). Similarly, we have presented both a base-specific chemical cleavage method, and an enzymatic method to identify the specific sequences of eluted oligonucleotides. Each approach meets different needs. The chemical cleavage method is more direct and cost-effective and is very appropriate for high complexity laboratories, or ultimately, closed systems. The enzymatic method described avoids the use of organic sequencing reagents, but has higher reagent costs. Because MASDA is modular, as instrumentation changes, the method used to identify the eluted oligonucleotide can change, without requiring changes in the remainder of the assay. Thus, each of the individual components of MASDA can be modified individually, without altering the basic protocol. These features are important in the clinical laboratory, where continual protocol changes are disruptive, and frequent purchase of capital equipment may not be possible.
Finally, because all sequence variants are analyzed using the same reaction conditions, assays for rare disorders can be analyzed in the same `run' as tests for common disorders. Thus, high volume economies of scale can be achieved for otherwise low volume tests. A more subtle benefit is the elimination of the internal support required to maintain multiple disease-specific protocols.
Currently, significant efforts are being made to develop informative databases on genotype-phenotype associations of existing and new mutations within known disease genes. In addition, there is an ever increasing interest in establishing the relationships between genotypes of patients involved in clinical trials and their response to various therapies. In addition to the diagnostic applications, MASDA will allow research laboratories to develop oligonucleotide libraries representative of previously identified expressed sequence tags or bi-allelic markers (polymorphisms) identified within the human genome. For this purpose, we currently are investigating the number of hybridized and eluted oligonucleotides that can be uniquely identified from any given positive sample. Given that the MASDA technology has the capacity to perform complex known mutation analysis on hundreds of patient samples with different disease indications in a single assay, we believe that it is suitable for immediate and future applications in both clinical and research laboratories.
When mutation-positive genomic DNA was not available, oligonucleotides representing 40 bp of endogenous gene sequence including the mutation were synthesized, cloned into pGEMR-3Zf(+) vectors (Promega Corporation, Madison, WI), and the presence of the mutation in each clone verified by sequencing (data not shown).
As a model system for complex mutation detection, mutations were selected from 33 regions in seven different genes. The genes included the CF transmembrane conductance regulator gene (CFTR), the [beta]-globin gene, the Tay-Sachs hexosaminidase gene (HEXA), the Gaucher gene (GCR), the Canavan aspartoacylase gene (ASPA), the breast cancer susceptibility gene (BRCA1) and the Fanconi anemia complementation group C gene (FACC).
PCR amplifications were performed using 1-2 [mu]g of genomic DNA or 10 ng of plasmid DNA in 100 [mu]l of reaction buffer containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 mM dNTPs and 0.05-0.1 U/ml of Taq polymerase (Perkin-Elmer, Norwalk, CT). For the different disease gene amplifications, the concentration of primers ranged from 0.2 to 1.6 [mu]M.
For DNA amplifications involving simultaneous multiplexes of three or more amplicons (CFTR 8-plex and 9-plex, ASPA 3-plex, BRCA1 4-plex and FACC 3-plex), the primers were chimeras of a sequence-specific region with a common `universal primer sequence' (UPS) as described by Shuber et al. (33 ). These primers facilitated rapid multiplex development and consistently robust amplifications. Primer sequences are not listed but may be furnished upon request.
DNA amplifications were performed using a Perkin-Elmer 9600 Thermal Cycler (Perkin-Elmer, Norwalk, CT). For CFTR, HEXA, ASPA, BRCA1 and FACC, the amplifications were carried out for 28 cycles with ramping (94oC/10 s hold with 48 s ramp, 60oC/10 s hold with 36 s ramp, 72oC/10 s hold with 38 s ramp) and a final 74oC hold for 5 min before cooling. For [beta]-globin and GCR, the amplification program consisted of 28 cycles with a 55oC anneal (94oC/10 s hold, 55oC/10 s hold, 74oC/10 s hold) and a final 74oC hold for 5 min before cooling.
Amplification products were analyzed by 2% agarose gel electrophoresis followed by ethidium bromide staining and visualization on a UV transilluminator (Fotodyne, New Berlin, WI).
Mutations from seven different genes were selected as candidates for a complex mutation detection assay. The 106 mutations examined included point mutations, deletions and insertions. Details of the selected mutations and gene amplifications are listed in Table 1 . References for any of the mutations will be provided upon request.
ASOs were 17mers synthesized and HPLC-purified by Operon Technologies (Alameda, CA). All oligonucleotides were quantitated by spectrophotometry and tested in independent hybridizations before being pooled. The sequences of the 106 different ASOs are not shown but may be provided upon request. Specified amounts of individual ASOs were combined into a pool of 106 ASOs so that the pool would contain the required amount of each specific ASO determined to be optimal for the pool hybridization. Aliquots of pooled ASOs were lyophilized and stored at -20oC.
Amplified products were denatured using 1.0 M NaOH, 2.0 M NaCl, 25 mM EDTA pH 8.0, containing bromophenol blue (30 [mu]l of 0.1% bromophenol blue/10 ml denaturant) for 5 min at room temperature. Denatured products were blotted onto Biotrans membrane (ICN Biomedicals Inc., Aurora, OH) using a 96-well format dot-blot apparatus (Life Technologies, Gaithersburg, MD). Membranes were neutralized in 2* SSC (0.15 M NaCl, 0.015 M trisodium citrate) for 5 min at room temperature and baked in a vacuum oven at 80oC for 15 min. Immediately before use, the membranes were rinsed in distilled water and placed in hybridization solution.
Aliquots of 106 pooled ASOs (one ASO sequence representing each mutation) were thawed, resuspended in distilled water and end-labeled in a single reaction containing 1* kinase buffer (New England Biolabs, Beverly, MA), 0.135 nmol of [[gamma]-32P]ATP (DuPont, Boston, MA) and 35 U of T4 polynucleotide kinase (New England Biolabs, Beverly, MA). The labeling reactions were incubated at 37oC for 1 h. The efficiency of the kinase reaction was monitored by chromatography on cellulose polyethyleneimine (PEI) plates (J.T. Baker Inc., Phillipsburg, NJ) using 0.75 M NaH2PO4 pH 3.5 buffer, followed by exposure of the plates to Kodak X-Omat X-Ray film (Eastman Kodak Company, Rochester, NY) at room temperature for 5 min.
Hybridization and wash conditions, using TMAC, were the same as previously described (25 ). For this protocol, the 96-well array of spotted genomic samples was marked with a grid so that positives identified in the hybridization could be located easily for the subsequent elution and ASO sequencing. Signal intensities generated from the different mutation-positive samples were optimized by adjusting the concentrations of each mutation-specific oligonucleotide within the hybridization. In order to achieve uniform hybridization signals, the final concentration of each labeled mutant ASO in the pool hybridization ranged from 0.008 to 1.8 pmol/ml, with the concentration of cold normal ASOs ranging from 0- to 200-fold excess of the corresponding mutant ASO.
To ensure that all 106 ASOs within the pool were labeled, membranes containing a positive control sample for each mutation were included in each hybridization.
Once washed, the blots were wrapped in plastic wrap and exposed to Kodak X-Omat X-Ray film (Eastman Kodak Company, Rochester, NY) at -80oC for 15 min to 1 h.
By chemical cleavage. The ASO hybridized to each mutation-positive sample was identified by eluting and sequencing the ASO. For a pool of 106 ASOs, sequencing `C' and `G' bases only was sufficient to identify the ASO sequence unambiguously and therefore allowed unequivocal identification of the corresponding mutation in the DNA sample.
The region of membrane containing each mutation-positive sample identified in the pool hybridization was excised, and the disc of Biotrans membrane placed in 100 [mu]l of distilled water and heated at 95oC for 10 min to elute the bound ASO. After cooling to room temperature, the membrane disc was discarded, and the eluted ASO was subjected to chemical sequencing.
Solid-phase chemical cleavage of the ASOs attached to a solid support was performed according to Rosenthal et al. (32 ) with minor changes. This method permitted simultaneous sequencing of all bound ASOs in a single reaction vessel. To attach the ASOs to a solid support prior to chemical cleavage, a small, labeled piece (6 mm * 3 mm) of CCS paper (32 ) was immersed in each tube containing eluted ASO, and incubated at 65oC for 1 h. All pieces of paper were then combined into a single 50 ml tube containing 25 ml of distilled water. The papers were then washed at room temperature three times (30 s/wash) with distilled water (25 ml/wash) followed by three washes (30 s/wash) with 96% ethanol (25 ml/wash). Papers with attached ASOs could be batch washed without cross-contamination (data not shown).
Once washed, the papers were air-dried and each piece cut into two, with one-third assigned for the `G' chemical cleavage reaction and two-thirds designated for the `C' cleavage reaction. All `C' reaction ASO solid supports were combined into one tube containing 1 ml of 4.0 M hydroxylamine HCl pH 6. For `G' reaction modifications, the combined pieces of paper were placed in 1 ml of 50 mM ammonium formate pH 3.5 and 7 [mu]l of dimethylsulfate added. Reactions were incubated at room temperature for 10 or 20 minutes for the `G' and `C' reaction respectively. Batch processing of >100 sequencing reactions was performed without cross-contamination of cleavage products (data not shown).
Washes were performed on the batch of `C' reaction papers and the batch of `G' reaction papers as described above for washes after attachment of the ASOs to the solid support. The papers were then air dried and each piece of paper placed in their designated location in a 96-well amplification tray (Perkin-Elmer, Norwalk, CT).
To cleave and elute the sequencing products off the solid support membrane, freshly prepared piperidine [50 [mu]l of 10% (v/v) piperidine] was added to each well, the tray was covered with a rubber gasket, and incubated at 90oC for 30 min in a thermal cycler.
For each cleavage reaction, the piperidine solution containing the eluted cleavage products was transferred to a fresh 96-well amplification tray and the piperidine evaporated for 2-3 h at room temperature. The evaporation step was repeated twice with 50% ethanol (35 [mu]l/well). ASO cleavage products were dissolved in 4 [mu]l of loading dye (90% formamide, 1* TBE, 0.1% bromophenol blue, 0.1% xylene cyanol) before gel electrophoretic resolution. Sequencing gels (20% polyacrylamide/8 M urea/TBE) were pre-run for 1 h at 2000 V, the samples were loaded, and electrophoresis continued until the bromophenol blue dye had migrated 14 cm from the origin. Sequencing gels were exposed to Kodak X-Omat X-ray film for 24-48 h.By enzymatic sequencing. Mutation-positive samples were identified, spots excised and ASOs eluted as described for the chemical cleavage method. The eluted samples were each placed in Microcon-10tm concentrators (Amicon Inc., Beverly, MA) and centrifuged at 14 000 r.p.m. for 15 min in a bench top microfuge. The eluates were then transferred to Microcon-3tm concentrators (Amicon Inc., Beverly, MA) and centrifuged at 14 000 r.p.m. for 30 min in a bench top microfuge. Both concentrators were washed twice with 100 [mu]l of distilled water per wash. (The Microcon-3tm concentrators were washed with the eluate from the corresponding Microcon-10tm concentrators.) The eluted ASOs were recovered from the top portion of the Microcon-3tm concentrator by three serial rinses with 20 [mu]l of distilled water/rinse, and the fractions pooled. The samples were lyophilized in a UniVapotm concentrator (Integrated Separation Systems, Natick, MA),re-dissolved in 5 [mu]l of distilled water, and used as sequencing primers in an enzymatic sequencing protocol designed to identify the eluted ASO. The sequencing reactions included a pool of oligonucleotide templates with each template (77mer) consisting of a 3' region (17 bp) as the primer-binding site uniquely complementary to a specific ASO, and a second unique region (17 bp) consisting of an `ASO-specific identifier sequence'. Sequencing products were only observed when an eluted ASO was bound to the complementary region of a unique template, acted as a primer and permitted cycle sequencing to reveal the identity of the downstream `ASO-specific identifier sequence'.
The cycle sequencing reactions contained a pool of ASO-specific templates (5 fmol/template), 0.5 [mu]l of Thermosequenase buffer concentrate (Amersham Life Science, Cleveland, OH), 0.125 [mu]l of Thermosequenase (32 U/[mu]l) and either `G' termination mix (15 [mu]M dATP, 15 [mu]M dCTP, 15 [mu]M dTTP, 15 [mu]M 7-deaza-dGTP and 4 [mu]M ddGTP) or `C' termination mix (15 [mu]M dATP, 15 [mu]M dGTP, 15 [mu]M dTTP, 15 [mu]M 7-deaza-dGTP and 4 [mu]M ddCTP) in a reaction volume of 8 [mu]l. Cycle sequencing was performed between 95oC for 30 s and 70oC for 1 min for 30 cycles, followed by a 2 min incubation at 70oC. Sequencing products were resolved on a 15% acrylamide/7 M urea gel before being exposed to Kodak X-Omat X-ray film at -70oC for ~16 h.
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*To whom correspondence should be addressed
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