DNA microarray Can be used to: Multiplex technology used in molecular biology and in medicine ■ measure changes in expression levels; ■ detect single nucleotide polymorphisms (SNP); ■ Arrayed series of thousands of microscopic spots of DNA oligonucleotides. ■ Probe target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets. ■ genotyping or in resequencing mutant genomes.
1. Sample preparation 2. Purification DNA microarray experiment The two samples to be compared (pairwise comparison) are grown/acquired. RNA DNA DNA/RNA bound to a protein The purified RNA is analysed for quality (by capillary electrophoresis) and quantity (by using a nanodrop spectrometer)
3. Reverse Transcription 4. Labelling DNA microarray experiment optional PCR amplification The label is added either in the RT step or in an additional step after amplification if present The labeled samples are then mixed with a propriety hybridization solution. SDS, SSC, dextran sulfate, a blocking agent, Denhardt's solution and formamine.
5. Hibridization 6. Scanning 7. Normalization and analysis DNA microarray experiment This mix is denatured and added to a pin hole in a microarray. The holes are sealed and the microarray hybridized. The microarray is dried and scanned in a special machine where a laser excites the dye and a detector measures its emission. After that the raw that is normalized for study
Uses and types Conditions 1. Gene expression Profiling The expression levels of thousands of genes are simultaneously monitored to study the effects of certain treatments, diseases , and developmental stages on gene expression. Genes How experimental conditions influenced production (expression) of mRNA for a set of genes. Green indicates reduced expression. Cluster analysis has placed a group of down regulated genes in the upper left corner.
Uses and types 1. Gene expression Profiling Requires the use of apropriete data sets. Ex: YEASTRACT And apropriete software. Ex: Genesis Dataset: Use apropriate public yeast dataset. Datasets represents the expression levels of genes, under differente conditions. Filtration: Sets the expression value of the genes within an interval. Normalization: Used to minimize errors during all the process
Uses and types 1. Gene expression Profiling Clustering of Genes. Clustering: Process of grouping a set of physical or abstract objects into classes of similar objects. Hierarchical: Organize elements into a tree, leaves represent genes and the length of the pathes between leaves represents the distances between genes. k-means: A specific number of clusters from a given set of genes. Its possible to associate a chosen cluster to its biological functions by comparison with datasets
Uses and types An SNP array is a useful tool to study the whole genome. 2. SNP detection Assessing genome content in different cells or closely related organisms Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, drugs, vaccines, and other agents. Hybridization-based methods
SNP microarrays Template DNA – Individual DNA, bigger than the probe. A C A C T A T G 3’ T C G Marqued dNTP’s C G A T Probe DNA 5’ The study heres is to know wether theres a variation of the first nucleotide thats not attached to the 3’ terminal of the probe.
Uses and types 3 - Real-time PCR Cells in all organisms regulate gene expression and turnover of gene transcripts Two common methods of quantification: ● use of fluorescent dyes; ● modified DNA oligonucleotide probes. Quantifying gene expression by traditional methods presents several problems. Firstly, detection of mRNA on a Northern blot or PCR products on a gel or Southern blot is time-consuming and does not allow precise quantification.
Uses and types 3 - Real-time PCR The TaqMan probe principle relies on the 5´–3´ nuclease activity of Taq polymerase. TaqMan probes are hydrolysis probes developed to increase the specificity of real-time PCR assays. Quantifying gene expression by traditional methods presents several problems. Northern blot or PCR products on a gel is time-consuming and does not allow precise quantification.
Conclusion Diagnostic of, for example, infectious diseases, cancer and genetic abnormalities. PCR used to provide quantitative measurements of gene transcription. Genetic expression of a particular gene changes over time Lab-On-A-Chip António Sousa 64427 MBioNano