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Dna Microarray
 

Dna Microarray

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    Dna Microarray Dna Microarray Presentation Transcript

    • Genomics
      DNA CHIps
      António Sousa
      64427 MBioNano
    • 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.
    • SNP microarrays
    • 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