microarrays.pptx molecular diagnostic by taimoor
- 2. DNA Microarray • DNA chip or biochip A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Used to measure/detect the expression levels of large numbers of genes simultaneously. • Gene expression analysis
- 3. Principle • Each spot contains Pico moles (10−12 moles) of a specific DNA sequence, known as probes. • Principle microarray a pattern of ssDNA probes immobilized of a microchip surface. • Probe-target hybridization is usually used to detect specific DNA or RNA in a sample.
- 4. Microarray chip Fig. 2 : DNA microarrays in medical practice Aitman et al., 2001 Fig. 1 : DNA probes attached in microarray features • solid-phase array (glass, plastic or silicon biochip) • Contain orderly microscopic "spots", called features
- 5. • Sample preparation and labelling • Hybridization • Washing • Image acquisition • Data analysis Fluorescent Dye Cyanine dye cy3 or cy5
- 7. • Fluorescently labeled target sequences that bind to a probe sequence generate a signal labeled cDNA with dye (cy3 or cy5) determine relative abundance of nucleic acid sequences in the target. • Total signal strength depend on Relative quantitation (the amount of target sample binding to the probes present on that spot).
- 8. Applications 1. Expression analysis or gene expression profiling. Measure changes in gene pattern of expression to better understand function of different genes. Response to pathogens or cancerous gene expression in infected to that in uninfected cells or tissues. 2. Functional genomics Identifying SNP among alleles within or between populations, genotyping
- 9. Cont.. 3. As a comparative genome tool (Analyze copy number variations (CNVs) in the DNA of a test sample compared to a reference sample) 4. Drug discovery Identifying drug-candidates measuring predisposition to disease Evaluating germline mutations in individuals or somatic mutations in cancers.
- 10. Cont.. 5. Identification of structural variations Identify changes in the genome resulting from some sort of mutation, deletion or repetition of genes or sequences .
- 11. Advantages • Provides data for thousand of genes • Fast and easy ( need single experiment instead of many). • Different parts of DNA can be used to study gene expression.
- 12. Protein microarray • High throughput method • Track activity of multiple proteins in cells, simultaneously Diagnostics • To discover new disease biomarkers • Monitoring the disease status • Response to therapies Protein expression profiling • Check the expression of protein in different cells, tissues e.g liver during the production of bile Protein – protein interaction
- 13. Protein – protein interaction • Expression of proteins involved in signal transduction and in many biological processes (including many diseases including Parkinson's disease and cancer).