Types of microarrays. Gene expression microarraysGenome Wide SNP and mutation analysis microarraysarray Comparative Genomic Hybridisation (aCGH)Array based Chromatin Immunoprecipitation Assays ( ChIP on chip)others: Sequencing ,Tiling . Gene expression microarrays consist of cDNA transcriptome probes which represent part of a particular gene. They give information about the relative differences in gene expression between two different cell populations eg treated cell compared to untreated c30279
2. Types of microarrays Gene expression microarrays
Genome Wide SNP and mutation analysis microarrays
array Comparative Genomic Hybridisation (aCGH)
Array based Chromatin Immunoprecipitation Assays ( ChIP on chip)
others: Sequencing ,Tiling
3. Gene expression microarrays consist of cDNA transcriptome probes which represent part of a particular gene.
They give information about the relative differences in gene expression between two different cell populations eg treated cell compared to untreated cell or cancer cells compared to normal cells.
The degree of up- and down-regulation can be estimated for each gene but not the amount (absolute number of molecules) of mRNA expressed in treated cells vs. untreated cells. Gene expression microarrays
4. Application With arrays capable of analysing the whole human transcriptome, gene expression analysis has been widely used for research on cell physiology and to find diagnostic markers / mechanisms for diseases such as cancer. Other applications include: determination of water pollution by examining expression profiles in mussels (exposed to different environmental condition), cell responses to irradiation (exposure to UV light).
Gene expression was and still is the most used application of microarray technology
5. Genome Wide SNP and mutation analysis microarrays. A SNP is typically a substitution of one base for another specific base. For example, a G is substituted with a C while all other bases in the close proximity of the SNP are unchanged. SNPs can be located inside as well as outside of genes. It is estimated that there are about 10 million single nucleotide polymorphisms (SNPs) in the human population. The SNPs are spread throughout the whole genome and can be used as genomic markers for finding links between genes and diseases. Two companies involve in designing SNPs microarrays are Affymetrix and Illumina. The technologies are based on allele-specific hybridisation and allele specific primer extension respectively.
6. Allele-specific hybridisation is based on probes that are centred over the mutation site so that the variant base is approximately in the middle of the probe. Centering the variant base to the middle of the probe destabilises mismatch hybrids maximally and the probe will therefore be highly sensitive to mutations in the target. At least two probes are used for detecting a particular SNP: one probe is perfectly matched with one allelic variant and another probe is specific for the other allelic variant. Though two probes suffice in principle, Affymetrix uses about 20 probes for each SNP analysed to obtain enough specificity in the assay.
7. Allele-specific primer extension is based on placing the probes so that the last nucleotide of the probe is that of a SNP. A polymerase reaction can be initiated if the probe ends with a perfect match while mismatch hybridisation will give a flapping 3’ end that cannot serve as an initiation structure for polymerisation.
8. The same technologies used for SNP genotyping can be used to genotype mutations that cause monogenetic diseases (sickle cell anaemia, phenylketonuria).
Uses include genotyping, forensic analysis, measuring predisposition to disease (tracking disease predisposition), identifying drug candidates, evaluating germline mutations in individuals or somatic mutations in cancers, assessing loss of heterozygosity or genetic linkage analysis.
9. array Comparative Genomic Hybridisation (aCGH) array Comparative Genomic Hybridisation or array CGH (aCGH) uses slides arrayed with small segments of DNA as the targets for analysis. These microarrays are created by the deposit and immobilisation of small amounts of DNA (known as probes) on a solid support in an ordered fashion. Probes vary in size from oligonucleotides manufactured to represent areas of interest (25-85 base pairs) to genomic clones such as bacterial artificial chromosomes (80,000 – 200,000 base pairs). The higher the number of base pair represented on the probes the less will be the resolution and the more likely of missing intervening regions.
10. In aCGH, the resolution is determined by the number of probes on the array. For example 32,000 different probes evenly distributed throughout the human genome gives array CGH a resolution of about 0.1M base . For example a microarray with probes selected from regions across the genome that are 1Mb apart will be unable to detect copy number changes of the intervening sequence.
More probes used the less will be the base pair in the probes
Resolution (in base pair) = Chromosome size / Number of probes.
aCGH are used to find large deletions and amplifications within genomes or copy number variation analysis (CNVs)
11. Array based Chromatin Immunoprecipitation Assays (ChIP on chip) Chromatin immunoprecipitation or ChIP assays are used to find the promoters that bind a specific transcription factor. DNA sequences bound to a particular protein can be isolated by immunoprecipitating that protein (ChIP), these fragments can then be hybridised to a microarray allowing the determination of protein binding site occupancy throughout the genome. The Chip microarray consists of probes towards the promoter regions. The Chip assay is not limited to transcription factors but can also be used for other DNA binding proteins such as histones.
12. Sequencing microarrays. Microarrays can be successfully used for resequencing purposes. Re-sequencing arrays are in principle the same as SNP array with the exception that four probes are used to determine the bases in a particular site. The variant base is centred in the middle of the probes. There are therefore four probes for each base investigated in a sequence and for example re-sequencing 10 bases in a row requires 40 probes. Therefore, sequencing of one million bases requires four million probes. Such re-sequencing can be used for identification of pathogens and mutational analysis of mitochondria and genetic variability of genome segments.
13. Tiling array. Genome tiling arrays consist of overlapping probes designed to represent a genomic region of interest, sometimes as large as an entire human chromosome The purpose is to empirically detect expression of transcripts or alternatively splice forms which may not have been previously know or predicted.