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DNA CHIPS
Genomics
António Sousa64427 MBioNano
DNA microarray
Multiplex technology used in molecular biology and in medicine
■ 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.
Can be used to:
■ measure changes in expression levels;
■ detect single nucleotide
polymorphisms (SNP); ■ 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.
RNADNADNA/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
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.
Conditions
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
Filtration:Sets the expression value of the genes within an interval.
Normalization:Used to minimize errors during all the process
Dataset:Use apropriate public yeast dataset. Datasets represents the expression levels of genes, under differente conditions.
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
2. SNP detection
Assessing genome content in different cells or closely related organisms
An SNP array is a useful tool to study the whole genome.
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
Molecular beacons
SNP microarrays
SNP microarrays
A
A
C
C
T
T
G
G
A
CTemplate DNA – Individual DNA, bigger than the probe.
5’
3’
Probe DNA
The study heres is to know wether theres a variation of the first nucleotide thats not attached to the 3’ terminal of the probe.
A
C
T
G
Marqued dNTP’s
T
SNP microarrays
Uses and types
3 - Real-time PCR
Two common methods of quantification:● use of fluorescent dyes;● modified DNA oligonucleotide probes.
Cells in all organisms regulate gene expression and turnover of gene transcripts
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
TaqMan probes are hydrolysis probes developed to increase the specificity of real-time PCR assays.
The TaqMan probe principle relies on the 5´–3´ nuclease activity of Taq polymerase.
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 Sousa64427 MBioNano
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