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