MSPC 233 Detection of Mutations and Bioinformatics PDF

Detection of mutations and Bioinformatics DR. BART DZUDZOR Detecting and Diagnosing Human Disease Conditions Detecting Genetic Diseases Detecting and Diagnosing Human Disease Conditions Molecular Diagnostics The use of DNA, RNA, and proteins to facilitate disease...

Detection of mutations and Bioinformatics DR. BART DZUDZOR Detecting and Diagnosing Human Disease Conditions Detecting Genetic Diseases Detecting and Diagnosing Human Disease Conditions Molecular Diagnostics The use of DNA, RNA, and proteins to facilitate disease detection, diagnosis, subclassification, prognosis, and monitoring response to therapy Detecting and Diagnosing Human Disease Conditions Advantages of Molecular Diagnostics Improvement in sensitivity High specificity Cost less Faster analysis time Detecting and Diagnosing Human Disease Conditions Detecting Genetic Diseases Fetal testing for chromosome abnormalities and defective genes Amniocentesis (Test at 16 weeks - karyotype) Chorionic villus sampling (Test at 8 to 10 weeks - karyotype) Detecting and Diagnosing Human Disease Conditions Detecting Genetic Diseases Testing for chromosome abnormalities and defective genes Fluorescence in situ hybridization (FISH) Fluorescence probes that are specific for chromosomes and/or genes Spectral karotype Molecular diagnosis of Sickle Cell Disease RFLP Direct Detection of a Sickle Cell Mutation by RFLP A specific hemoglobin mutation Wild Type Mutant Pro Glu Pro Val CCT GAG CCT GTG [DdeI cuts at CTNAG] DdeI site no DdeI site AS AS SS AA Gene encoding sickle cell b-subunit Gene encoding Wild type b-subunit Detecting and Diagnosing Human Disease Conditions Hybridization - single-stranded oligonucleotides are permitted to interact so that complexes, or hybrids, are formed by molecules with sufficiently similar, complementary sequences Target - the nucleotide sequence the oligonucleotide is designed to hybridize with Probe - the nucleic acid that carries a marker for detection Making SNPs Make Sense Detecting and Diagnosing Human Disease Conditions Dot Blots Assay for detecting SNPs Uses PCR amplified DNA blotted onto a membrane Unbound ASO probe is washed off Bound ASO probe is detected by radioactive or colorimetric assays Dot Blot animation Detecting and Diagnosing Human Disease Conditions Allele-Specific Oligonucleotide (ASO) Dot Blot to detect Sickle Cell Anemia Detecting and Diagnosing Human Disease Conditions Reverse Dot Blot Instead of binding DNA to the membrane, an array of ASOs are bound to a membrane and hybridized to labeled target DNA Reverse Dot Blot video Detecting and Diagnosing Human Disease Conditions Detecting Genetic Diseases Single Nucleotide Polymorphisms (SNPs) One of the most common forms of genetic variation Estimated that one SNP occurs approximately every 1,000-3,000 bp in the human genome 99.9 percent of the DNA sequence will be exactly the same –> 80% of 0.1 percent variation will be SNPs Most have no effect because they occur in non-protein coding regions (introns) 10 pharmaceuticals donated millions in a collaborative partnership called the SNP Consortium Medical Products and Applications of Biotechnology Microarray technology Can compare levels of gene expression in different tissues Applications in cancer research Microarray animation Detecting and Diagnosing Human Disease Conditions Microarray A chip containing thousands of pieces of single stranded DNA molecules DNA is isolated from a patient, fluorescently labeled, and hybridized to the microarray A laser scanner measures the intensity of the fluorescence to indicate the binding of the patients DNA to the SNP or gene on the microarray Detecting and Diagnosing Human Disease Conditions Detecting Genetic Diseases Identifying sets of disease genes by microarray analysis Microarray created with known diseased genes or SNPs DNA from a patient is tagged with fluorescent dyes and then hybridized to the chip Binding of a patient’s DNA to a gene sequence on the chip indicates that the person’s DNA has a particular mutation or SNP Introduction to Bioinformatics Dr. Bart Dzudzor Know where to get information Genome-wide Association Studies (GWAS) xxare