How can Microarrays be used to determine copy number variants and SNPS?

Question

I need to know the ways to determine CNVs using microarrays and how to determine SNPs using microarrays

Answer 1

A DNA microarray is a high-throughput technology used in molecular biology and in medicine. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles of a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target.

In standard microarrays, the probes are attached to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide or others). The solid surface can be glass or a silicon chip, in which case they are commonly known as gene chip or colloquially Affy chip when an Affymetrix chip is used. Other microarray platforms, such as Illumina, use microscopic beads, instead of the large solid support. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system.

DNA microarrays can be used to measure changes in expression levels or to detect single nucleotide polymorphisms (SNPs) (see Types of arrays section). Microarrays also differ in fabrication, workings, accuracy, efficiency, and cost (see fabrication section). Additional factors for microarray experiments are the experimental design and the methods of analyzing the data (see Bioinformatics section).

For a detailed explanation of how a microarray experiment is done, see DNA microarray experiment.

Contents [hide]

1 History

2 Uses and types

2.1 Gene expression profiling

2.2 Comparative genomic hybridization

2.3 SNP detection

2.4 Chromatin immunoprecipitation on Chip

2.5 Genotyping

2.6 Resequencing

2.7 Tiling

3 Fabrication

3.1 Spotted vs. oligonucleotide arrays

3.2 Two-channel vs. one-channel detection

4 Microarrays and bioinformatics

4.1 Experimental Design

4.2 Standardization

4.3 Statistical analysis

4.4 Relation between probe and gene

5 Public databases of microarray data

6 Online microarray data-analysis programs and tools

7 See also

8 References

9 Glossary

10 External links



[edit] History

Microarray technology evolved from Southern blotting, where fragmented DNA is attached to a substrate and then probed with a known gene or fragment. The use of a collection of distinct DNAs in arrays for expression profiling was first described in 1987, and the arrayed DNAs were used to identify genes whose expression is modulated by interferon.[1] These early gene arrays were made by spotting cDNAs onto filter paper with a pin-spotting device. The use of miniaturized microarrays for gene expression profiling was first reported in 1995, [2] and a complete eukaryotic genome (Saccharomyces cerevisiae) on a microarray was published in 1997. [3]

[edit] Uses and types



Two Affymetrix chipsArrays of DNA can be spatially arranged, as in the commonly known gene chip (also called genome chip, DNA chip or gene array), or can be specific DNA sequences labelled such that they can be independently identified in solution. The traditional solid-phase array is a collection of microscopic DNA spots attached to a solid surface, such as glass, plastic or silicon biochip. The affixed DNA segments are known as probes (although some sources use different terms such as reporters). Thousands of them can be placed in known locations on a single DNA microarray.

DNA microarrays can be used to detect DNA (as in comparative genomic hybridization), or detect of RNA (most commonly as cDNA after reverse transcription) that may or may not be translated into proteins. The process of measuring gene expression via cDNA is called expression analysis or expression profiling.

Since an array can contain tens of thousands of probes, a microarray experiment can accomplish that many genetic tests in parallel. Therefore arrays have dramatically accelerated many types of investigation. Applications include:

[edit] Gene expression profiling

Main article: expression profiling

In an mRNA or gene expression profiling experiment the expression levels of thousands of genes are simultaneously monitored to study the effects of certain treatments, diseases, and developmental stages on gene expression. For example, microarray-based gene expression profiling can be used to identify genes whose expression is changed in response to pathogens or other organisms by comparing gene expression in infected to that in uninfected cells or tissues. [4]

[edit] Comparative genomic hybridization

Main article: Comparative genomic hybridization

Assessing genome content in different cells or closely related organisms. [5] [6]

[edit] SNP detection

Main article: SNP array

Identifying single nucleotide polymorphism among alleles within or between populations.[7]

[edit] Chromatin immunoprecipitation on Chip

Main article: ChIP-on-chip

DNA sequences bound to a particular protein can be isolated by imunoprecipitating that protein (ChIP), these fragments can be then hybridized to a microarray (such as a tiling array) allowing the determination of protein binding site occupancy throughout the genome. Example protein to imunoprecipitate are histone modifications (H3K27me3, H3K4me2, H3K9me3, etc), Polycomb-group protein (PRC2:Suz12, PRC1:YY1) and trithorax-group protein (Ash1) to study the epigenetic landscape or RNA Polymerase II to study the transcription lanscape.

[edit] Genotyping

Main article: SNP array

DNA microarrays can also be used to scan the entire sequence of a genome to identify genetic variation at certain locations.

SNP microarrays are a type of DNA microarray that are used to identify genetic variation in individuals and across populations. [7] Short oligonucleotide arrays can be used to identify single nucleotide polymorphisms (SNPs) responsible for genetic variation and the potential source of susceptibility to genetically caused diseases. Generally termed genotyping applications, DNA microarrays may be used in this fashion for forensic applications, genotyping, rapidly discovering or measuring genetic predisposition to disease, or identifying DNA-based drug candidates.

These SNP microarrays are also being used to profile somatic mutations in cancer, specifically loss of heterozygosity events and amplifications and deletions of regions of DNA. Amplifications and deletions can also be detected using comparative genomic hybridization (CGH) in conjunction with microarrays, but may be limited in detecting novel Copy Number Polymorphisms, or CNPs, by probe coverage.

[edit] Resequencing

Resequencing arrays have been developed to sequence portions of the genome in individuals. These arrays may be used to evaluate germline mutations in individuals, or somatic mutations in cancers.[citation needed]

[edit] Tiling

Genome tiling arrays include overlapping oligonucleotides designed to cover an entire genomic region of interest. Many companies have successfully designed tiling arrays that cover whole human chromosomes.

[edit] Fabrication

Microarrays can be manufactured in different ways, depending on the number of probes under examination, costs, customization requirements, and the type of scientific question being asked. Arrays may have as few as 10 probes to up to 390,000 micron-scale probes from commercial vendors.

[edit] Spotted vs. oligonucleotide arrays

A DNA microarray being created

A DNA microarray being printed by a robot at the University of Delaware. • File format: Ogg• File size: 5.05 MB• Duration: 1m11s

  



Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, [8] or electrochemistry on microelectrode arrays.

In spotted microarrays, the probes are oligonucleotides, cDNA or small fragments of PCR products that correspond to mRNAs. The probes are synthesized prior to deposition on the array surface and are then "spotted" onto glass. A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface. The resulting "grid" of probes represents the nucleic acid profiles of the prepared probes and is ready to receive complementary cDNA or cRNA "targets" derived from experimental or clinical samples. This technique is used by research scientists around the world to produce "in-house" printed microarrays from their own labs. These arrays may be easily customized for each experiment, because researchers can choose the probes and printing locations on the arrays, synthesize the probes in their own lab (or collaborating facility), and spot the arrays. They can then generate their own labeled samples for hybridization, hybridize the samples to the array, and finally scan the arrays with their own equipment. This provides a relatively low-cost microarray that is customized for each study, and avoids the costs of purchasing often more expensive commercial arrays that may represent vast numbers of genes that are not of interest to the investigator. Publications exist which indicate in-house spotted microarrays may not provide the same level of sensitivity compared to commercial oligonucleotide arrays, [9] possibly owing to the small batch sizes and reduced printing efficiencies when compared to industrial manufactures of oligo arrays. Applied Microarrays offers a commercial array platform called the "CodeLink" system where 30-mer oligonucleotide probes (sequences of 30 nucleotides in length) are piezoe