PUBLICATIONS

Recent Publications co-authored by Asper Biotech's Scientists: 

1. Genotyping microarray as a novel approach for the detection of ATP7B gene mutations in patients with Wilson disease
   Gojova L., Jansova E., Külm M., Pouchla S., Kozak L.
   Clinical Genetics 2008: 73: 441–452
   
   Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism that is caused by mutations in the ATP7B gene. To date, more than 300 mutations have been described in this gene. Molecular diagnostics of WD utilizes restriction enzyme digestion, multiplex ligation-dependent probe amplification or a direct sequencing of the whole gene. To simplify and speed up the screening of ATP7B mutations, we have developed a genotyping microarray for the simultaneous detection of 87 mutations and 17 polymorphisms in the ATP7B gene based on the arrayed primer extension reaction. The patient’s DNA is amplified in four multiplex polymerase chain reactions, fragmented products are annealed to arrayed primers spotted on a chip, which enables DNA polymerase extension reactions with fluorescently labeled dideoxynucleotides. The Wilson microarray was validated by screening 97 previously genetically confirmed WD patients. In total, we detected 43 mutations and 15 polymorphisms that represent a majority of the common mutations occurring in the Czech and Slovak populations. All screened sequence variants were detected with 100% accuracy. The Wilson chip appears to be a rapid, sensitive and cost-effective tool, representing the prototype of a disease chip that facilitates and speeds up the screening of potential WD patients.
   
   
2. Microarray-based mutation analysis of the ABCA4 gene in Spanish patients with Stargardt disease: evidence of a prevalent mutated allele
   Diana Valverde, R. Riveiro-Alvarez, Sara Bernal, Kaie Jaakson, Montserrat Baiget, Rafael Navarro, Carmen Ayuso
   Molecular Vision 2006; 12:902-908

   Purpose: To evaluate, in a pool of affected families, the mutation spectrum in Stargardt patients from Spain, using the ABCR400 microarray that contains described sequence variants in the gene encoding for the photoreceptor specific ATP-binding cassette transporter (ABCA4).
   Methods: We analyzed 76 Spanish patients with STGD1 for a population-specific survey on the sequence variations in the ABCA4 gene, using the ABCR400 microarray.
   Results: Potential disease-associated alleles were identified in 91 of the 152 STGD1 chromosomes studied, resulting in a detection rate of 60%. The two mutant alleles were found in 33/76 patients (43%), whereas in 25/76 cases (33%) only one allele could be identified. In the remaining 18 patients no mutations were found. In total, we identified 40 sequence variations that could be related to the disease. The vast majority of these substitutions (35/40) were missense mutations. Three frameshift mutations and two splicing variants were also found.
   Conclusions: We identified a major disease-associated allele, R1129L, which accounted for 24% of the mutated alleles detected, and a high frequency (12%) of complex alleles.
    

3. Comprehensive Arrayed Primer Extension Array for the Detection of 59 Sequence Variants in 15 Conditions Prevalent Among the (Ashkenazi) Jewish Population.
   Iris Schrijver, Maigi Külm, Phyllis I. Gardner, Eugene P. Pergament, and Morris B. Fiddle
   Journal of Molecular Diagnostics, Vol. 9, No. 2, April 2007
   
   In the Ashkenazi Jewish population, serious and lethal genetic conditions occur with relatively high frequency. A single test that encompasses the majority of population-specific mutations is not currently available. For comprehensive carrier screening and molecular diagnostic purposes, we developed a population-specific and inclusive microarray. The arrayed primer extension genotyping microarray carries 59 sequence variant detection sites, of which 53 are detectable bi-directionally. These sites represent the most common variants in Tay-Sachs disease, Bloom syndrome, Canavan disease, Niemann-Pick A, familial dysautonomia, torsion dystonia, mucolipidosis type IV, Fanconi anemia, Gaucher disease, factor XI deficiency, glycogen storage disease type 1a, maple
   syrup urine disease, nonsyndromic sensorineural hearing loss, familial Mediterranean fever, and glycogen storage disease type III. Several mutations in the selected disorders that are not prevalent per se in the Ashkenazi Jewish populations, as well pseudodeficiency
   alleles, are also included in the array. The initial technical evaluation of this microarray demonstrates that it is comprehensive, robust, sensitive, specific , and easily modifiable. This cost-effective array is based on a diversely applied platform technology and is suitable for both carrier screening and disease detection in Ashkenazi and Sephardic Jewish populations.
    

4. Association study of sporadic Parkinson's disease genetic risk factors in patients from Russia by APEX technology.
   Shadrina M, Nikopensius T, Slominsky P, Illarioshkin S, Bagyeva G, Markova E, Ivanova-Smolenskaia I, Kurg A, Limborska S, Metspalu A.
   Neurosci Lett. 2006 Sep 25;405(3):212-6. Epub 2006 Jul 28.

   Most patients with Parkinson’s disease (PD) have sporadic form of the disease with a multifactorial etiology due to interactions between environmental conditions and the genetic constitution of the individuals.We have analyzed by APEX technology 50 single nucleotide polymorphisms (SNPs) in 19 genes related to cholecystokinin, serotonin, dopamine and opioid neurotransmission. Significant differences in the allele and genotype frequencies between the controls and PD patients were detected for four SNPs from three genes (serotonin 2A receptor (rs6311, P = 0.043), Wolfram syndrome 1 (rs1801211, P = 0.007), proopiomelanocortin (rs28930368, P = 0.026 and rs2071345, P = 0.027) genes). Two SNPs in proopiomelanocortin (POMC) gene were also associated with different clinical forms of PD. Our data suggest that at least three genes involved in neurotransmitter systems may have more specific role in genetic predisposition to PD.

    

5. Simultaneous multigene mutation detection in patients with sensorineural hearing loss through a novel diagnostic microarray: a new approach for newborn screening follow-up.
   Gardner P, Oitmaa E, Messner A, Hoefsloot L, Metspalu A, Schrijver I.
   Pediatrics. 2006 Sep;118(3):985-94.
   
   The advent of universal newborn hearing screening in the United States and other countries, together with the identification of genes involved in the process of hearing, have led to an increase in both the need and opportunity for accurate molecular diagnosis of patients with hearing loss. Deafness and hearing impairment have a genetic cause in at least half the cases. The molecular genetic basis for the majority of these patients remains obscure, however, because of the absence of associated clinical features in approximately 70% (ie, nonsyndromic hearing loss) of patients, genetic heterogeneity, and the lack of molecular genetic tests that can evaluate a large number of mutations across multiple genes. DESIGN: We report on the development of a diagnostic panel with 198 mutations underlying sensorineural (mostly nonsyndromic) hearing loss. This panel, developed on a microarray, is capable of simultaneous evaluation of multiple mutations in 8 genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5 and the mitochondrial genes encoding 12S rRNA and tRNA-Ser[UCN]). RESULTS: The arrayed primer extension array for sensorineural hearing loss is based on a versatile platform technology and is a robust, cost-effective, and easily modifiable assay. Because hearing loss is a major public health concern and common at all ages, this test is suitable for follow-up after newborn hearing screening and for the detection of a genetic etiology in older children and adults. CONCLUSIONS: Comprehensive and relatively inexpensive genetic testing for sensorineural hearing loss will improve medical management for affected individuals and genetic counseling for their families.
     

6. Development of a Genotyping Microarray for Usher Syndrome
   Cremers FP, Kimberling WJ, Kulm M, de Brouwer A, van Wijk E, Te Brinke H, Cremers CW, Hoefsloot LH, Banfi S, Simonelli F, Fleischhauer JC, Berger W, Kelley PM, Haralambous E, Bitner-Glindzicz M, Webster AR, Saihan Z, Debaere E, Leroy BP, Silvestri G, McKay G, Koenekoop RK, Millan JM, Rosenberg T, Joensuu T, Sankila EM, Weil D, Weston MD, Wissinger B, Kremer H.
   J Med Genet. 2006 Sep 8
   
   Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, 8 genes have been implicated, which together comprise 347 protein-coding exons. Therefore, sequence analysis and the most routinely used mutation scanning techniques are not cost-effective for molecular diagnostics of Usher syndrome. To improve DNA-diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. METHODS: Allele-specific oligonucleotides corresponding to 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. The accuracy of the microarray was analysed using DNAs from 158 patients with known mutations; the efficiency of the microarray was analysed using DNAs from 370 novel European and American patients with Usher syndrome. RESULTS: Validation of the microarray yielded an accuracy of >98%. Among the novel patients, sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I (USH1), 45/189 (24%) patients with Usher syndrome type II (USH2), 6/21 (29%) patients with Usher syndrome type III (USH3), and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. DISCUSSION: The Usher genotyping microarray represents a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.
     

7. A Quality Assessment Survey of SNP Genotyping Laboratories
   Lahermo P, Liljedahl U, Alnaes G, Axelsson T, Brookes A, Ellonen P, Groop P-E, Halldén C, Holmberg D, Holmberg K, Keinänen M, Kepp K, Kere J, Kiviluoma P, Kristensen V, Lindgren C, Odeberg J, Osterman P, Parkkonen M, Saarela J, Sterner M, Strömqvist L, Talas U, Wessman M, Palotie A, Syvänen A-C (2006) A Quality Assessment Survey of SNP Genotyping Laboratories. Hum Mutat, 27(7):711-714
   
   To survey the quality of SNP genotyping, a joint Nordic quality assessment (QA) round was organized between 11 laboratories in the Nordic and Baltic countries. The QA round involved blinded genotyping of 47 DNA samples for 18 or six randomly selected SNPs. The methods used by the participating laboratories included all major platforms for small- to medium-size SNP genotyping. The laboratories used their standard procedures for SNP assay design, genotyping, and quality control. Based on the joint results from all laboratories, a consensus genotype for each DNA sample and SNP was determined by the coordinator of the survey, and the results from each laboratory were compared to this genotype. The overall genotyping accuracy achieved in the survey was excellent. Six laboratories delivered genotype data that were in full agreement with the consensus genotype. The average accuracy per SNP varied from 99.1 to 100% between the laboratories, and it was frequently 100% for the majority of the assays for which SNP genotypes were reported. Lessons from the survey are that special attention should be given to the quality of the DNA samples prior to genotyping, and that a conservative approach for calling the genotypes should be used to achieve a high accuracy.
     
    

8. Evaluation of arrayed primer extension for TP53 mutation detection in breast and ovarian carcinomas.
   Pedro Kringen, Anna Bergamaschi, Eldri Undlien Due, Yun Wang, Elda Tagliabue, Jahn M. Nesland, Aune Ahman, Neeme Tönisson, and Anne-Lise Børresen-Dale.
   BioTechniques, vol. 39 , no 5 (2005): pp 755-761
   

   Mutations in the tumor suppressor gene TP53 are associated with a wide range of different cancers and may have prognostic and therapeutic implications. Methods for rapid and sensitive detection of mutations in this gene are therefore required. In order to make screening more effective, a commercially available TP53 genotyping microarray from Asper Biotech has been constructed by arrayed primer extension (APEX). The present study is the first report that blindly evaluates the efficiency of the second generation APEX TP53 genotype chip outside the Asper laboratory and compares it to temporal temperature gradient electrophoresis (TTGE) and sequencing of TP53 for mutation detection in ovarian and breast cancer samples. All nucleotides in the TP53 gene from exon 2–9 are included on the chip by synthesis and application of sequence-specific oligonucleotides. The chip was validated by screening 48 breast and 11 ovarian cancer cases, all of which had previously been analyzed by TTGE and sequencing. APEX scored 17 of 20 sequence variants, missing one deletion, one insertion, and a missense mutation. Resequencing efficiency using APEX was 92% for both DNA strands and 99.5% for sense and/or antisense strand. We conclude that the APEX TP53 microarray is a robust, rapid, and comprehensive screening tool for sequence alterations in tumors.

    

9. Genotyping microarray (disease chip) for leber congenital amaurosis: detection of modifier alleles.
   
   Zernant J, Kulm M, Dharmaraj S, den Hollander AI, Perrault I, Preising MN, Lorenz B, Kaplan J, Cremers FP, Maumenee I, Koenekoop RK, Allikmets R.
   Invest Ophthalmol Vis Sci. 2005 Sep;46(9):3052-9.
   
   PURPOSE: Leber congenital amaurosis (LCA) is an early-onset inherited disorder of childhood blindness characterized by visual impairment noted soon after birth. Variants in at least six genes (AIPL1, CRB1, CRX, GUCY2D, RPE65, and RPGRIP1) have been associated with a diagnosis consistent with LCA or early-onset retinitis pigmentosa (RP). Genetically heterogeneous inheritance complicates the analyses of LCA cases, especially in patients without a family history of the disorder, and conventional methods are of limited value. METHODS: To overcome these limitations, arrayed primer extension (APEX) technology was used to design a genotyping microarray for early-onset, severe retinal degenerations that includes all of the >300 disease-associated variants currently described in eight genes (in addition to the six just listed, the early-onset RP genes LRAT and MERTK were added). The resultant LCA array allows simultaneous detection of all known disease-associated alleles in any patient with early-onset RP. The array was validated by screening 93 confirmed patients with LCA who had known mutations. Subsequently, 205 novel LCA cases were screened on the array, followed by segregation analyses in families, if applicable. RESULTS: The microarray was >99% effective in determining the existing genetic variation and yielded at least one disease-associated allele in approximately one third of the novel patients. More than two (expected) variants were discovered in a substantial fraction (22/300) of the patients, suggesting a modifier effect from more than one gene. In support of the latter hypothesis, the third allele segregated with a more severe disease phenotype in at least five families. CONCLUSIONS: The LCA genotyping microarray is a robust and cost-effective screening tool, representing the prototype of a disease chip for genotyping patients with a genetically heterogeneous condition. Simultaneous screening for all known LCA-associated variants in large LCA cohorts allows systematic detection and analysis of genetic variation, facilitating prospective diagnosis and ultimately predicting disease progression.
   

10. Genotyping Microarray for the Detection of More Than 200 CFTR Mutations in Ethnically Diverse Populations
    
    Schrijver I, Oitmaa E, Metspalu A, Gardner P. 
    J Mol Diagn. 2005 Aug;7(3):375-87

    Cystic fibrosis (CF), which is due to mutations in the cystic fibrosis transmembrane conductance regulator gene, is a common life-shortening disease. Although CF occurs with the highest incidence in Caucasians, it also occurs in other ethnicities with variable frequency. Recent national guidelines suggest that all couples contemplating pregnancy should be informed of molecular screening for CF carrier status for purposes of genetic counseling. Commercially available CF carrier screening panels offer a limited panel of mutations, however, making them insufficiently sensitive for certain groups within an ethnically diverse population. This discrepancy is even more pronounced when such carrier screening panels are used for diagnostic purposes. By means of arrayed primer extension technology, we have designed a genotyping microarray with 204 probe sites for CF transmembrane conductance regulator gene mutation detection. The arrayed primer extension array, based on a platform technology for disease detection with multiple applications, is a robust, cost-effective, and easily modifiable assay suitable for CF carrier screening and disease detection.
    

     

11. Arrayed Primer Extension Resequencing of Mutations in the TP53 Tumor Suppressor Gene: Comparison with Denaturing HPLC and Direct Sequencing.

      
    Le Calvez F, Ahman A, Tonisson N, Lambert J, Temam S, Brennan P, Zaridze DG, Metspalu A, Hainaut P.
    Clin Chem. 2005 Jul;51(7):1284-7.
       

    Mutations of TP53 (17p13.1; OMIM 191170; PubMed accession number X54156) are common in cancers and are typically missense within exons 4–9, impairing the capacity of p53 to transactivate genes involved in cell cycle arrest, apoptosis, and DNA repair (1). Functionally, mutations may differ according to their nature and position, as well as to the presence of a common polymorphism at codon 72 (arginine or a proline) in the mutant allele (2). Knowing TP53 mutation status has potential applications for cancer prognosis (3)(4) and early diagnosis (5), identification of mutagen "fingerprints" (1)(6), and prediction of therapeutic outcomes (7)(8). To achieve this purpose, sensitive, fast, and cost-effective methods are needed to assess the whole coding sequence plus exon/intron boundaries. Current approaches are based on mutation prescreening with single strand conformational polymorphism analysis, temporal temperature gradient electrophoresis, or denaturing HPLC (DHPLC) combined with direct sequencing of relevant PCR fragments [reviewed in Ref. (9)]. These methods are labor-intensive, difficult to standardize, and in some cases, of limited sensitivity. In recent years, 2 microarray methods for resequencing TP53 have been described: the Affymetrix p53 GeneChip array, described elsewhere (10)(11), and the Arrayed Primer Extension (APEX), based on incorporation of 4 dye terminators into oligonucleotide primers that each identify a base in the target sequence (12). In 2002, we described an APEX array for resequencing TP53 exons 2–9, which contain 95% of known mutations in TP53 (13). Here we compare the sensitivity and detection limits of APEX with a standard method, DHPLC/direct sequencing, and discuss the potential of APEX for application to cancer diagnostic or prognostic purposes.

      

12. Genotyping Microarray (Gene Chip) for the ABCR (ABCA4) Gene
    

    K. Jaakson, J. Zernant, M. Kulm, A. Hutchinson, N. Tonisson, D. Glavac¡, M. Ravnik-Glavac¡, M. Hawlina, M.R. Meltzer, R.C. Caruso, F. Testa, A. Maugeri, C.B. Hoyng, P. Gouras, F. Simonelli, R.A. Lewis, J.R. Lupski, F.P.M. Cremers, and R. Allikmets
    Hum Mutat 22:395–403, 2003.

    Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all B400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides.We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research.
      

13. Reliable Detection of beeta-Thalasseemia and G6PDMutations by a DNA Microarray.
    
    Gemignani, F., Perra, C., Landi, St., Canzian, F., Kurg, A., Tõnisson, N., Galanello, R., Cao, A., Metspalu, A. and Romeo, G.
    Clinical Chemistry 48, No.11, 2002 2051-2054
    
        

14. A first-generation linkage disequilibrium map of human chromosome 22.
       
    Dawson, E., Abecasis, G.R, Bumpstead, S., Chen, Y., Hunt, S., Beare, D.M., Pabial, J., Dibling, T., Tinsley, E., Kirby, S., Carter, D., Papaspyridonos, M., Livingstone, S., Ganske, R., Lõhmussaar, E., Zernant, J., Tõnisson, N., Remm, M., Mägi, R., Puurand, T., Vilo, V., Kurg, A., Rice, K., Deloukas, P., Mott, R., Metspalu, A., Bentley, D. R., Cardon, L.R. and Dunham, I.
    Nature 418, 544-548, August 2002
    
    DNA sequence variants in specific genes or regions of the human genome are responsible for a variety of phenotypes such as disease risk or variable grug response. These variants can be investigated directly, or through their non-random associations with neighbouring markers (called linkage disequilibrium (LD)). Here are report measurement of LD along the complete sequence of human chromosome 22. Duplicate genotyping and analysis of 1,504 markers in Centre d’Etude du Polymorphisme Humain (CEPH) reference families at a median spacing of 15 kilobases (kb) reveals a highly variable pattern of LD along the chromosome, in whichextensive regions of nearly complete LD up to 804 kb in length are interspersed with regions of little or no detectable LD. The LD patterns are replicated in a panel of unrelated UK Caucasians. There is a strong correlation between high LD and low recombination frequency in the extant genetic map, suggesting that historical and contemporary recombination rates are similar. This study demostrates the feasibility of developing genome-wide maps of LD. 
         

15. Evaluating the arrayed primer extension resequencing assay of TP53 tumor suppressor gene.
        
    Neeme Tõnisson, Jana Zernant, Ants Kurg, Hendrik Pavel, Georg Slavin, Hanno Roomere, Aune Meiel, Pierre Hainaut and Andres Metspalu
    Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, Issue 8, 5503-5508.
    
    Identification of mutations in the tumor suppressor gene TP53 has implications for the molecular epidemiology and for the molecular pathology of human cancer. We have developed and evaluated an arrayed primer extension assay for covering both strands of a region of the coding sequence containing more than 95% of the mutations described so far in TP53. On average, 97.5% of the arrayed TP53 gene sequence can be analyzed from either sense or antisense strands, and 81% from both strands. A patient DNA sample is amplified and annealed to arrayed primers, which then promote DNA polymerase extension reactions with four fluorescently labeled dideoxynucleotides. The TP53 gene chip spans exons 2–9 plus two introns from both strands. The performance of the assay was evaluated by using freshly extracted genomic DNA, as well as DNA extracted from archival (paraffin-embedded) DNA samples. The arrayed primer extension-based TP53 gene test provides an accurate and efficient tool for DNA sequence analysis of this frequently mutated gene for both research and clinical applications. 
       

16. Primer extension from two-dimensional oligonucleotide grids for DNA sequence analysis. 
    
    Metspalu A, Saulep H, Kurg A, Tõnisson N. 
    Genomis: Commercial Opportunities from a Scientific Revolution (ed. by Dixon GK, Copping LG, Livingstone D), BIOS Scientific Publishers 1998:217-219.
      
17. Arrayed primer extension on the DNA chip – method and applications. 
    
    Tõnisson N, Kurg A, Lõhmussaar E, Metspalu A. 
    Microarray Biochip Technology (2000), 247-263 Biotechniques Books (ed. by Mark Schena), Eaton Publishing 2000
      
18. Laser diagnostic system for rapid mutation identification. .
    
    Kurg A, Tõnisson N, Metspalu A, Berik E. 
    Medical and Biological Engineering and Computing 37, suppl. 1 (1999), 311
      

19. Arrayed Primer Extension: Solid phase four-color DNA resequencing and mutation detection technology. 
    
    Kurg A, Tõnisson N, Tollett J, Georgiou I, Shumaker J, Metspalu A. 
    Genetic Testing Vol.4 #1, 1-7
    
    The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging and data analysis. The method is based upon array of oligonucleotides, immobilized via 5’ end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides.  A mutation will be detected by a change in the color code of the primer sites. The technology was applied for analyzing of 10 common ß-thalassemia mutations. Nine patient DNA samples each of which carries a different mutation and four wild-type DNA samples were correctly identified. The signal to noise ratio of this technology is on the average 40:1 which enables the identification of heterozygous mutations with high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.
      

20. Unravelling genetic data by arrayed primer extension.
    
    Tõnisson N, Kurg A, Kaasik K, Lõhmussaar E, Metspalu A. 
    Clin. Chem. Lab. Med. 2000; 38(2): 165-170

    We have developed a method for arrayed primer extension (APEX) on an oligonucleotide microchip together with the 4-color fluoresence imaging equipment and supporting software, that allows analysis of the DNA sequence and changes in it. Mutation analysis of BRCA1 gene and single nucleotide polymorphism (SNP) chip for genotyping were used as a model system.

    Chip surface chemistry, template preparation and APEX reaction conditions were optimised and the assay is ready to be implemented in variety of DNA analysis from SNP testing to DNA resequencing.
       

21. Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays.
    
    Pastinen T, Kurg A, Metspalu A, Peltonen L, Syvanen AC.
    Genome Res 1997 Jun;7(6):606-14
    
    We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.

Publications, which involve Asper's genotyping technology

1. Microarray-Based Mutation Detection and Phenotypic Characterization of Patients with Leber Congenital Amaurosis

   Suzanne Yzer, Bart P. Leroy, Elfride De Baere,Thomy J. de Ravel, Marijke N. Zonneveld, Krysta Voesenek, Ulrich Kellner, Jose P. Martinez Ciriano, Jan-Tjeerd H. N. de Faber, Klaus Rohrschneider, Ronald Roepman, Anneke I. den Hollander, Johannes R. Cruysberg, Franc¸oise Meire, Ingele Casteels, Norka G. van Moll-Ramirez, Rando Allikmets, L. Ingeborgh van den Born, and Frans P. M. Cremers
   Vis Sci. 2006;47:1167–1176) DOI:10.1167/iovs.05-0848
     

2. Microarray-based mutation analysis of the ABCA4 (ABCR) gene in autosomal recessive cone-rod dystrophy and retinitis pigmentosa. 
     
   Klevering BJ, Yzer S, Rohrschneider K, Zonneveld M, Allikmets R, van den Born LI, Maugeri A, Hoyng CB, Cremers FP.
   European Journal of Human Genetics (2004) 12, 1024–1032.
     

3. Evaluation of a microarray for genotyping polymorphisms related to xenobiotic metobism and DNA repair.
     
   Landi, S., Gemignani, F., Gioia-Patricola, L., Chabrier, A., Canzian, F.
   BioTechniques, Vol. 35, No.4 (2003), 816-827.
     

4. Optical microarray biosensing techniques.
   
   Bally M., Halter M., Vörös J., Grandin M.
   Surface and Interface Analysis 2006, 38: 1442- 1458
   
   

5. Clinical and Molecular Genetics of Leber's Congenital Amaurosis: A Multicenter Study of Italian Patients
   
   Francesca Simonelli, Carmela Ziviello, Francesco Testa, Settimio Rossi, Elisa Fazzi, Paolo Emilio Bianchi, Maurizio Fossarello, Sabrina Signorini, Chiara Bertone, Silvana Galantuomo, Francesco Brancati, Enza Maria Valente, alfredo Ciccodicola, Ernesto Rinaldi, Alberto Auricchio and Sandro Banfi
   IOVS , September 2007, Vol. 48, No.9