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PubMed2003

[DNA chips for gene expression analysis and their application in diagnostics].

Due to large genome projects more and more genetic information and more and more data about genetic background of various diseases can be obtained. By means of microarrays thousands to hundreds of thousands of molecular analysis can be performed at a time, rapidly, under identical conditions, hereby microarrays provide a solution to continuously increasing throughput demands from large genomic, pharmacogenetic and proteomic programmes. Microarrays offer unprecedented opportunities for qualitative and quantitative analysis of gene expression, understanding gene function and detection gene alterations, polimorphisms. Microarray expression profiles will define stages of different tumor types and new prognostic subgroups in cancer and other diseases. For individual patients, more precise diagnosis and risk assessment based on expression profiles are already achievable, leading to more accurate determination of prognosis and more individually tailored treatment. Microarrays will play an important role in diagnostics of infectious diseases and identification of novel diagnostic and therapeutic targets. Wide-spread application of microarrays in clinical practice is not typical today, but their production on an industrial scale and the increased information provided by microarrays should assure their entry into clinical practice as very powerful, sensitive, specific and rapid tools of diagnostics of various diseases.

Communicable Diseases↗
PubMed2002

Genome structures embossed by oligonucleotide-stickiness.

MOTIVATION: An unmanageably large amount of data on genome sequences is accumulating, prompting researchers to develop new methods to analyze them. We have devised a novel method designated oligostickiness, a measure roughly proportional to the binding affinity of an oligonucleotide to a DNA of interest, in order to analyze genome sequences as a whole. RESULTS: Fifteen representative genomes such as Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, H. sapiens and others were analyzed by this method using more than 50 probe dodecanucleotides, offering the following findings: (i) Genome sequences can be specifically featured by way of oligostickiness maps. (ii) Oligostickiness analysis, which is similar to but more informative than (G + C) content or repetitive sequence analysis, can reveal intra-genomic structures such as mosaic structures (E. coli and B. subtilis) and highly sticky/non-sticky regions of biological meanings. (iii) Some probe oligonucleotides such as dC(12) and dT(12) can be used for classifying genomes, clearly discriminating prokaryotes and eukaryotes. (iv) Based on global oligostickiness, which is the average value of the local oligostickinesses, the features of a genome could be visualized in spider web mode. The pattern of a spider web as well as a set of oligostickiness maps is highly characteristic to each genome or chromosome. Thus, we called it as chromosome texture, leading to a finding that all the chromosomes contained in a cell, so far investigated, have a common texture. AVAILABILITY: Oligostickinesses maps used in this work are available at http://gp.fms.saitama-u.ac.jp/ CONTACT: koichi@fms.saitama-u.ac.jp

Algorithms↗
PubMed2003

A microarray to analyze methylation patterns of p16(Ink4a) gene 5'-CpG islands.

OBJECTIVES: Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation on the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput approach for analyzing methylation patterns. The aim of this study was to develop a new method to analyze methylation patterns of p16(Ink4a) gene. DESIGN AND METHODS: We selected a 336 bp segment of the 5' untranslated region and the first exon of the p16(Ink4a) gene, as the target sequence, which include the most densely packed CpG fragment of the islands containing 32 CpG sites. A set of oligonucleotide probes was designed to assemble a DNA microarray to discriminate the methylation patterns of several adjacent CpG sites. RESULTS: Methylation patterns of human p16(Ink4a) gene were mapped and the results were validated by bisulphite DNA sequencing. A good reproducibility was observed in several parallel experiments. CONCLUSIONS: The methylation oligonucleotide microarray can be applied as a useful and powerful tool to map methylation patterns in multiple CpG island sites.

Base Sequence↗
PubMed2006

Free energy of DNA duplex formation on short oligonucleotide microarrays.

DNA/DNA duplex formation is the basic mechanism that is used in genome tiling arrays and SNP arrays manufactured by Affymetrix. However, detailed knowledge of the physical process is still lacking. In this study, we show a free energy analysis of DNA/DNA duplex formation these arrays based on the positional-dependent nearest-neighbor (PDNN) model, which was developed previously for describing DNA/RNA duplex formation on expression microarrays. Our results showed that the two ends of a probe contribute less to the stability of the duplexes and that there is a microarray surface effect on binding affinities. We also showed that free energy cost of a single mismatch depends on the bases adjacent to the mismatch site and obtained a comprehensive table of the cost of a single mismatch under all possible combination of adjacent bases. The mismatch costs were found to be correlated with those determined in aqueous solution. We further demonstrate that the DNA copy number estimated from the SNP array correlates negatively with the target length; this is presumably caused by inefficient PCR amplification for long fragments. These results provide important insights into the molecular mechanisms of microarray technology and have implications for microarray design and the interpretation of observed data.

Base Pair Mismatch↗
PubMed2003

Microarray as a model for quantitative visualization chemistry.

For visualization of proteins or nucleic acids, direct and indirect in situ fluorescence and absorption methods (immunohistochemistry and cytochemistry) have existed for many years. The authors describe a new experimental approach using microarray as a model to quantitatively compare both visualization methods. The spots obtained with the microarray robot had a progressive twofold decrease in concentrations and are used as objects with known amounts of DNA. Subsequent hybridization resulted in a direct fluorescence (DF) label or in hapten for indirect fluorescence (IF) and absorption. The results show that the image of the object in the IF method is larger than that in the DF method because of an edge effect, with stronger staining at the circumference. This leads to a higher plateau level and an 8- to 10-fold reduction in the detection threshold for IF compared with DF. These features are especially useful for one-color DNA-related microarray analysis, such as single nucleotide polymorphism, loss of heterozygosity, and mutation analysis, provided that the spots are not designed directly adjacent to each other, so that the edge effect is taken into account. The slope of the linear range for the IF method is much steeper than for the DF method, pointing to a narrow dynamic range in immunohistochemistry. It is noteworthy that the detection limit for absorption images after indirect immunoenzyme visualization is lower than for the DF images. The indirect immunohistochemistry semiquantitative absorption signal was at least similar compared with the DF fluorescence. In conclusion, an explanation for the difficulties experienced in quantitative immunohistochemistry is provided, and the data emphasize that in general, for daily pathology, semiquantitative patterns should suffice. Indirect labeling of DNA has useful characteristics for application in microarray analyses because of the large signal enhancement.

Carbocyanines↗
PubMed2003

The use of genetic microarray analysis to classify and predict prognosis in haematological malignancies.

The introduction of microarrays offers the opportunity to examine the expression of many thousands of genes in a single experiment. Investigations in leukaemia and lymphoma have led to the identification of a number of subgroups, with a defined gene expression pattern, not previously identified by morphology, cytogenetics or molecular techniques. In many cases these expression patterns can be linked to the tumour cells normal developmental counterpart, and represent distinct disease subgroups with different clinical presentations and outcomes. The technology has also identified genes that may be important in tumour cell biology including key genes in cell proliferation, adhesion, apoptosis, and the development of drug resistance. These early studies demonstrate that genetic microarrays will be useful in classifying haematological malignancies, predicting response to treatment, predicting prognosis, and identifying novel targets for therapy.

Computational Biology↗
PubMed2001

Identification of rifampin-resistant Mycobacterium tuberculosis strains by hybridization, PCR, and ligase detection reaction on oligonucleotide microchips.

Three new molecular approaches were developed to identify drug-resistant strains of Mycobacterium tuberculosis using biochips with oligonucleotides immobilized in polyacrylamide gel pads. These approaches are significantly faster than traditional bacteriological methods. All three approaches-hybridization, PCR, and ligase detection reaction--were designed to analyze an 81-bp fragment of the gene rpoB encoding the beta-subunit of RNA polymerase, where most known mutations of rifampin resistance are located. The call set for hybridization analysis consisted of 42 immobilized oligonucleotides and enabled us to identify 30 mutant variants of the rpoB gene within 24 h. These variants are found in 95% of all mutants whose rifampin resistance is caused by mutations in the 81-bp fragment. Using the second approach, allele-specific on-chip PCR, it was possible to directly identify mutations in clinical samples within 1.5 h. The third approach, on-chip ligase detection reaction, was sensitive enough to reveal rifampin-resistant strains in a model mixture containing 1% of resistant and 99% of susceptible bacteria. This level of sensitivity is comparable to that from the determination of M. tuberculosis drug resistance by using standard bacteriological tests.

Antibiotics, Antitubercular↗
PubMed2005

Secondary structure in the target as a confounding factor in synthetic oligomer microarray design.

BACKGROUND: Secondary structure in the target is a property not usually considered in software applications for design of optimal custom oligonucleotide probes. It is frequently assumed that eliminating self-complementarity, or screening for secondary structure in the probe, is sufficient to avoid interference with hybridization by stable secondary structures in the probe binding site. Prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from Brucella suis 1330 demonstrates that the properties of the target molecule have the potential to strongly influence the rate and extent of hybridization between transcript and tethered oligonucleotide probe in a microarray experiment. RESULTS: Despite the relatively high hybridization temperatures and 1M monovalent salt imposed in the modeling process to approximate hybridization conditions used in the laboratory, we find that parts of the target molecules are likely to be inaccessible to intermolecular hybridization due to the formation of stable intramolecular secondary structure. For example, at 65 degrees C, 28 +/- 7% of the average cDNA target sequence is predicted to be inaccessible to hybridization. We also analyzed the specific binding sites of a set of 70mer probes previously designed for Brucella using a freely available oligo design software package. 21 +/- 13% of the nucleotides in each probe binding site are within a double-stranded structure in over half of the folds predicted for the cDNA target at 65 degrees C. The intramolecular structures formed are more stable and extensive when an RNA target is modeled rather than cDNA. When random shearing of the target is modeled for fragments of 200, 100 and 50 nt, an overall destabilization of secondary structure is predicted, but shearing does not eliminate secondary structure. CONCLUSION: Secondary structure in the target is pervasive, and a significant fraction of the target is found in double stranded conformations even at high temperature. Stable structure in the target has the potential to interfere with hybridization and should be a factor in interpretation of microarray results, as well as an explicit criterion in array design. Inclusion of this property in an oligonucleotide design procedure would change the definition of an optimal oligonucleotide significantly.

Bacterial Proteins↗
PubMed2003

Expression profiling identifies genes that continue to respond to insulin in adipocytes made insulin-resistant by treatment with tumor necrosis factor-alpha.

We have employed microarray technology using RNA from normal 3T3-L1 adipocytes and from 3T3-L1 adipocytes made insulin-resistant by treatment with tumor necrosis factor-alpha to identify a new class of insulin-responsive genes. These genes continued to respond normally to insulin even though the adipocytes themselves were metabolically insulin-resistant, i.e. they displayed a significantly decreased rate of insulin-stimulated glucose uptake. Approximately 12,000 genes/expressed sequence tags (ESTs) were screened. Of these, 40 genes/ESTs were identified that became insulin-resistant as expected (e.g. Socs-3, junB, and matrix metalloproteinase-11). However, 61 genes/ESTs continued to respond normally to insulin. Although some of these genes were previously shown to be regulated by insulin (e.g. Glut-1 and beta3-adrenergic receptor), other novel insulin-sensitive genes were also identified (e.g. Egr-1, epiregulin, Fra-1, and ABCA1). Real-time reverse transcription-PCR analysis confirmed the expression patterns of several of the differentially expressed genes. One gene that remained insulin-sensitive in the insulin-resistant adipocytes is the transcription factor Egr-1. Using an antisense strategy, we show that tissue factor and macrophage colony-stimulating factor, two cardiovascular risk factors, are downstream EGR-1 target genes in the adipocyte. Taken together, these data support the hypothesis that some signaling pathways remain insulin-sensitive in metabolically insulin-resistant adipocytes. These pathways may promote abnormal gene expression in hyperinsulinemic states like obesity and type II diabetes and thus may contribute to pathologies associated with these conditions.

3T3-L1 Cells↗
PubMed2002

Ontogeny and the possible function of a novel epidermal growth factor-like repeat domain-containing protein, NELL2, in the rat brain.

In this study we investigated the mRNA expression of NELL2, a neural tissue-specific epidermal growth factor (EGF)-like repeat domain-containing protein, in the developing and adult rat CNS using in situ hybridization histochemistry and northern blot analysis. The possible candidates that interact with or be regulated by NELL2 were screened with a cDNA expression array in antisense (AS) NELL2 oligodeoxynucleotide (ODN)-injected rat hypothalami. NELL2 mRNA was detected as early as embryonic day 10, and was predominant in the CNS throughout the pre-natal stages. Its expression gradually increased during embryonic development and its strong expression was observed throughout the CNS until embryonic day 20. It was detected in the ventricular zone of the spinal cord, medulla and pons in 12-day-old-embryos, suggesting that NELL2 plays a role in the neurogenesis of these areas. After birth its expression gradually decreased, but high levels of expression could be observed in the tenia tecta, piriform cortex, hippocampus, dentate gyrus, cerebellar cortex, ambiguus nucleus, and inferior olivary nucleus of adult rat brains. The analysis of cDNA expression arrays revealed that the administration of AS NELL2 ODN markedly decreased the expression of several Ca2+-binding proteins and those involved in the transport and release of vesicles such as EF-hand Ca2+-binding protein p22 and rab7. This finding was confirmed by relative reverse transcription-polymerase chain reaction. The effect of NELL2 on synaptic vesicle content in median eminence (ME) nerve terminals was determined with synaptophysin levels as a marker protein in the AS NELL2 ODN-injected rat. It was significantly decreased by the AS ODN. These data suggest that NELL2 may play an important role in the development of the CNS as well as maintenance of neural functions, by regulating the intracellular machinery involving Ca2+ signaling, synaptic transport and/or release of vesicles.

Animals↗
PubMed2005

Precision profiling and components of variability analysis for Affymetrix microarray assays run in a clinical context.

Although gene expression profiling using microarray technology is widely used in research environments, adoption of microarray testing in clinical laboratories is currently limited. In an attempt to determine how such assays would perform in a clinical laboratory, we evaluated the analytical variability of Affymetrix microarray probesets using two generations of human Affymetrix chips (U95Av2 and U133A). The study was designed to mimic potential clinical applications by using multiple operators, machines, and reagent lots, and by performing analyses throughout a period of several months. A mixed model analysis was used to evaluate the relative contributions of multiple factors to overall variability, including operator, instrument, run, cRNA/cDNA synthesis, and changes in reagent lots. Under these conditions, the average probeset coefficient of variation (CV) was relatively low for present probesets on both generations of chips (mean coefficient of variation, 21.9% and 27.2% for U95Av2 and U133A chips, respectively). The largest contribution to overall variation was chip-to-chip (residual) variability, which was responsible for between 40 to 60% of the total variability observed. Changes in individual reagent lots and instrumentation contributed very little to the overall variability. We conclude that the approach demonstrated here could be applied to clinical validation of Affymetrix-based assays and that the analytical precision of this technique is sufficient to answer many biological questions.

DNA, Complementary↗
PubMed2003

Use of DNA array to screen blastocyst genes potentially involved in the process of murine implantation.

Conceptus implantation to the mother's uterus is a complex series of events involving coordinated expression of numerous genes at both the embryonic and the uterine sides. Since there are no suitable in vivo or in vitro experimental models, sequential changes occurring during the peri-implantation periods have not been well characterized. Using GeneChip technology and a recently introduced murine in vitro model of implantation, the expression of embryonic genes was examined before and after attachment to the uterine stromal cells. Instead of RNA or mRNA, amplified cRNA was subjected to the GeneChip analysis because amounts of mRNA in each blastocyst were minimal. Among 6,500 gene transcripts examined, changes in mRNA levels for 802 genes were identified. Of these detections, transcripts previously unsuspected were changes in a group of tumor suppressor and stress-induced genes, whose transcripts increased as embryos attached to the membrane. Validity of the data was evaluated using reverse transcription-polymerase chain reaction and in situ hybridization analyses, both of which confirmed developmental changes in selected gene expressions during pre- and post-attachment periods. The present data suggest that GeneChip technology would be very useful for finding genes previously unsuspected, and this method should be used as an initial step, particularly as a screening tool, toward the dissection of complex mechanisms such as the processes of implantation.

Animals↗
PubMed2006

Inhibitive effects of anti-oxidative vitamins on mannitol-induced apoptosis of vascular endothelial cells.

OBJECTIVE: Study blood vessel injury and gene expression indicating vascular endothelial cell apoptosis induced by mannitol with and without administration of anti-oxidative vitamins. METHODS: Healthy rabbits were randomly divided into four groups. Mannitol was injected into the vein of the rabbit ear in each animal. Pre-treatment prior to mannitol injection was performed with normal saline (group B), vitamin C (group C) and vitamin E (group D). Blood vessel injury was assessed under electron and light microscopy. In a second experiment, cell culture specimen of human umbilical vein endothelial cells were treated with mannitol. Pre-treatment was done with normal saline (sample B), vitamin C (sample C) and vitamin E (sample D). Total RNA was extracted with the original single step procedure, followed by hybridisation and analysis of gene expression. RESULTS: In the animal experiment, serious blood vessel injury was seen in group A and group B. Group D showed light injury only, and normal tissue without pathological changes was seen in group C. Of all 330 apoptosis-related genes analysed in human cell culture specimen, no significant difference was seen after pre-treatment with normal saline, compared with the gene chip without pre-treatment. On the gene chip pre-treated with vitamin C, 45 apoptosis genes were down-regulated and 34 anti-apoptosis genes were up-regulated. Pre-treatment with vitamin E resulted in the down-regulation of 3 apoptosis genes. CONCLUSION: Vitamin C can protect vascular endothelial cells from mannitol-induced injury.

Animals↗
PubMed2000

Global analysis of herpes simplex virus type 1 transcription using an oligonucleotide-based DNA microarray.

More than 100 transcripts of various abundances and kinetic classes are expressed during phases of productive and latent infections by herpes simplex virus (HSV) type 1. To carry out rapid global analysis of variations in such patterns as a function of perturbation of viral regulatory genes and cell differentiation, we have made DNA microchips containing sets of 75-mer oligonucleotides specific for individual viral transcripts. About half of these are unique for single transcripts, while others function for overlapping ones. We have also included probes for 57 human genes known to be involved in some aspect of stress response. The chips efficiently detect all viral transcripts, and analysis of those abundant under various conditions of infection demonstrates excellent correlation with known kinetics of mRNA accumulation. Further, quantitative sensitivity is high. We have further applied global analysis of transcription to an investigation of mRNA populations in cells infected with a mutant virus in which the essential immediate-early alpha27 (U(L)54) gene has been functionally deleted. Transcripts expressed at 6 h following infection with this mutant can be classified into three groups: those whose abundance is augmented (mainly immediate-early transcripts) or unaltered, those whose abundance is somewhat reduced, and those where there is a significant reduction in transcript levels. These do not conform to any particular kinetic class. Interestingly, levels of many cellular transcripts surveyed are increased. The high proportion of such transcripts suggests that the alpha27 gene plays a major role in the early decline in cellular gene expression so characteristic of HSV infection.

Animals↗
PubMed2004

Mechanical stretch is a highly selective regulator of gene expression in human bladder smooth muscle cells.

Application of mechanical stimuli has been shown to alter gene expression in bladder smooth muscle cells (SMC). To date, only a limited number of "stretch-responsive" genes in this cell type have been reported. We employed oligonucleotide arrays to identify stretch-sensitive genes in primary culture human bladder SMC subjected to repetitive mechanical stimulation for 4 h. Differential gene expression between stretched and nonstretched cells was assessed using Significance Analysis of Microarrays (SAM). Expression of 20 out of 11,731 expressed genes ( approximately 0.17%) was altered >2-fold following stretch, with 19 genes induced and one gene (FGF-9) repressed. Using real-time RT-PCR, we tested independently the responsiveness of 15 genes to stretch and to platelet-derived growth factor-BB (PDGF-BB), another hypertrophic stimulus for bladder SMC. In response to both stimuli, expression of 13 genes increased, 1 gene (FGF-9) decreased, and 1 gene was unchanged. Six transcripts (HB-EGF, BMP-2, COX-2, LIF, PAR-2, and FGF-9) were evaluated using an ex vivo rat model of bladder distension. HB-EGF, BMP-2, COX-2, LIF, and PAR-2 increased with bladder stretch ex vivo, whereas FGF-9 decreased, consistent with expression changes observed in vitro. In silico analysis of microarray data using the FIRED algorithm identified c-jun, AP-1, ATF-2, and neurofibromin-1 (NF-1) as potential transcriptional mediators of stretch signals. Furthermore, the promoters of 9 of 13 stretch-responsive genes contained AP-1 binding sites. These observations identify stretch as a highly selective regulator of gene expression in bladder SMC. Moreover, they suggest that mechanical and growth factor signals converge on common transcriptional regulators that include members of the AP-1 family.

Algorithms↗
PubMed2004

Identification of the CREB-binding protein/p300-interacting protein CITED2 as a peroxisome proliferator-activated receptor alpha coregulator.

Like other nuclear receptors, the peroxisome proliferator-activated receptors (PPARs) use a wide variety of protein-protein interactions to properly regulate transcription of target genes. In an attempt to identify novel PPAR-interacting proteins, a cDNA expression library was screened with bacterially expressed PPARalpha. One of the genes identified as a PPARalpha-associated protein by interaction cloning was the CREB-binding protein/p300-interacting transactivator with ED-rich tail 2 (CITED2, also called p35srj/mrg1/msg1). This coactivator interacted directly with PPARalpha in the presence or absence of ligand predominantly via the ligand binding domain of the nuclear receptor. In transient transfection reporter assays, CITED2 acted as a dose-dependent coactivator of PPARalpha-dependent transcriptional regulation in the presence of several exogenous ligands. CITED2 also increased PPARgamma-dependent regulation of reporter genes but had no effect on PPARbeta activity. To determine whether CITED2 affects endogenous gene expression, this protein was stably overexpressed (CITED2+) or repressed by small inhibitor RNA (CITED2-) in immortalized mouse hepatocytes. Relative to the control stably transfected or CITED2-cells, CITED2+ cells had an increased rate of cell proliferation. Microarray analysis and real time PCR showed that several genes are differentially affected by PPARalpha ligands in CITED2+ versus CITED2-cells. Genes that were affected by PPARalpha ligands in a CITED2-modulatory manner include angiopoietin-like protein 4, forkhead C2, hypoxia-inducible factor-1alpha, and MAPK phosphatase 1. Interestingly these genes share common functions in that they are known to promote vascularization and angiogenesis in response to hypoxia. The results described here suggest that CIT-ED2 is a coactivator of PPARalpha and that both proteins may participate in signaling cascades of hypoxic response and angiogenesis.

3T3-L1 Cells↗
PubMed2005

The expanding role of microarrays in the investigation of macrophage responses to pathogens.

In the last few years, microarray technology has emerged as the method of choice for large-scale gene expression studies. It provides an efficient and rapid method to investigate the entire transcriptome of a cell. No research field has benefited more from microarray technology than the study of the exquisite interplay between pathogens and hosts. Numerous microarray studies have now been published in this field, which have provided insights into the mechanisms of host defence and the tactics employed by pathogens to circumvent these protection strategies. These studies have led to a more comprehensive understanding of the host immune response and identified new avenues of research for potential control strategies against pathogens. In the past, research has concentrated on human and mouse microarrays to investigate host-pathogen interactions, regardless of the host species. This trend is changing with the ever-expanding sequence resources now available for many pathogen and host species, including livestock animals. The use of species-specific microarrays has furthered our understanding of host-pathogen interactions for particular organisms and aided in the annotation of unknown genes. Macrophages play a central role in the host's innate and adaptive immune responses to pathogens. These cells are in the first line of defence and interact with a wide range of pathogens; many of which have evolved strategies to circumvent the macrophage defence mechanisms and survive within these cells. In this report, we review the wealth of studies using microarray technology to investigate the response of macrophages to pathogens. These studies illustrate how microarray technology has expanded our understanding of the dialogue between macrophage and pathogen and provide examples of the benefits and pitfalls of using this technique. Furthermore, we discuss the resources available to use microarray analysis to study the immune response of a non-human, non-rodent species, the cow.

Animals↗
PubMed2004

Microarray-based AMASE as a novel approach for mutation detection.

Alterations in the p53 tumor suppressor gene are important events in many cases of human cancers. We have developed a novel microarray based approach for re-sequencing and mutation detection of the p53 gene. The method facilitates rapid and simple scanning of the target gene sequence and could be expanded to include other candidate cancer genes. The methodology employs the previously described apyrase-mediated allele-specific extension reaction (AMASE). In order to re-sequence the selected region, four extension oligonucleotides with different 3'-termini were used for each base position and they were covalently attached to the glass slide's surface. The amplified single-stranded DNA templates were then hybridized to the array followed by in situ extension with fluorescently labeled dNTPs in the presence of apyrase. The model system used was based on analysis of a 15 bp stretch in exon 5 of the p53 gene. Mutations were scored as allelic fractions calculated as (wt)/(wt + mut) signals. When apyrase was included in the extension reactions of wild type templates, the mean allelic fraction was 0.96. When apyrase was excluded with the same wild type templates, significantly lower allelic fractions were obtained. Two 60-mer synthetic oligonucleotides were used to establish the detectable amount of mutations with AMASE and a clear distinction between all the points could be made. Several samples from different stages of skin malignancies were also analyzed. The results from this study imply the possibility to efficiently and accurately re-sequence the entire p53 gene with AMASE technology.

Alleles↗