Polymerase chain reaction (PCR) and sequence specific oligonucleotide probes (SSOP) genotyping assay for detection of genes associated with rheumatoid arthritis and multiple sclerosis. 2010

Konstantina Nikolaou, and Fanis G Kalatzis, and Nikolaos Giannakeas, and Sofia Markoula, and Anthi Chatzikyriakidou, and Ioannis Georgiou, and Dimitrios I Fotiadis
Unit of Medical Technology and Intelligent Information Systems, Dept. of Material Science & Engineering, University of Ioannina, GR-45110, Greece. konikol@cc.uoi.gr

In this paper an assay for the detection of genes associated with rheumatoid arthritis (RA) and multiple sclerosis, using polymerase chain reaction (PCR) and sequence specific oligonucleotide probes (SSOP) is presented, in order to be further applied in a portable Lab-On-Chip (LOC) device. A substantial part of these reagents were based on the literature (11th International Histocompatibility Workshop, IHW), bearing the advantage of proven successful implementation in genotyping, while others were designed for this study. More precisely, our methodology discriminates HLA-DRB1 as DRB1*01, *04 and *10, which include shared epitope (SE) alleles associated with RA and additionally DRB1*15 allele, including DRB1*1501 associated with MS (broad genotyping method). To further present the basic elements of the assay for high resolution genotyping of SE DRB1 alleles, we provide as an example the case of HLA-DRB1*10 alleles (HLADRB1* 100101, *100102, *100103, *1002 and *1003). Regarding the methodology for developing a detection assay, for SNPs associated with RA or MS the basic steps are presented. DNA sequence data are obtained from IMGT/HLA and SNP database. Online software tools are used to define hybridization specificity of primers and probes towards human DNA, leading to hybridization patterns that uniquely designate a target allele and evaluate parameters influencing PCR efficiency. Respecting current technological limitations of autonomous molecular-based LOC systems the approach of broad genotyping of HLA-DRB1*01/*04/*10/*15 genes, is intended to be initially used, leaving, high resolution genotyping of SE alleles for future implementations. This method is easy to be updated and extended to detect additional associated loci with RA or MS.

UI MeSH Term Description Entries
D009103 Multiple Sclerosis An autoimmune disorder mainly affecting young adults and characterized by destruction of myelin in the central nervous system. Pathologic findings include multiple sharply demarcated areas of demyelination throughout the white matter of the central nervous system. Clinical manifestations include visual loss, extra-ocular movement disorders, paresthesias, loss of sensation, weakness, dysarthria, spasticity, ataxia, and bladder dysfunction. The usual pattern is one of recurrent attacks followed by partial recovery (see MULTIPLE SCLEROSIS, RELAPSING-REMITTING), but acute fulminating and chronic progressive forms (see MULTIPLE SCLEROSIS, CHRONIC PROGRESSIVE) also occur. (Adams et al., Principles of Neurology, 6th ed, p903) MS (Multiple Sclerosis),Multiple Sclerosis, Acute Fulminating,Sclerosis, Disseminated,Disseminated Sclerosis,Sclerosis, Multiple
D005838 Genotype The genetic constitution of the individual, comprising the ALLELES present at each GENETIC LOCUS. Genogroup,Genogroups,Genotypes
D006684 HLA-DR Antigens A subclass of HLA-D antigens that consist of alpha and beta chains. The inheritance of HLA-DR antigens differs from that of the HLA-DQ ANTIGENS and HLA-DP ANTIGENS. HLA-DR,Antigens, HLA-DR,HLA DR Antigens
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000483 Alleles Variant forms of the same gene, occupying the same locus on homologous CHROMOSOMES, and governing the variants in production of the same gene product. Allelomorphs,Allele,Allelomorph
D001172 Arthritis, Rheumatoid A chronic systemic disease, primarily of the joints, marked by inflammatory changes in the synovial membranes and articular structures, widespread fibrinoid degeneration of the collagen fibers in mesenchymal tissues, and by atrophy and rarefaction of bony structures. Etiology is unknown, but autoimmune mechanisms have been implicated. Rheumatoid Arthritis
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D015345 Oligonucleotide Probes Synthetic or natural oligonucleotides used in hybridization studies in order to identify and study specific nucleic acid fragments, e.g., DNA segments near or within a specific gene locus or gene. The probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the probe include the radioisotope labels 32P and 125I and the chemical label biotin. Oligodeoxyribonucleotide Probes,Oligonucleotide Probe,Oligoribonucleotide Probes,Probe, Oligonucleotide,Probes, Oligodeoxyribonucleotide,Probes, Oligonucleotide,Probes, Oligoribonucleotide
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D059811 HLA-DRB1 Chains A subtype of HLA-DRB beta chains that includes over one hundred allele variants. The HLA-DRB1 subtype is associated with several of the HLA-DR SEROLOGICAL SUBTYPES. HLA DRB1,HLA-DRB1,HLA-DRB1 Antigen,Antigen, HLA-DRB1,Chains, HLA-DRB1,HLA DRB1 Antigen,HLA DRB1 Chains

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