DNA Microarray- Definition, Principle, Procedure, Types

DNA microarrays are powerful tools that allow researchers to measure the expression levels of thousands of genes simultaneously. They can also be used to detect genetic variations, such as single nucleotide polymorphisms (SNPs), copy number variations (CNVs), and insertions or deletions (indels).

A DNA microarray consists of a solid surface, such as a glass slide or a silicon chip, to which thousands of DNA probes are attached. Each probe is a short DNA sequence that is complementary to a specific target sequence in the sample. The sample can be either genomic DNA or complementary DNA (cDNA) derived from messenger RNA (mRNA).

The sample is labeled with a fluorescent dye and hybridized to the microarray under specific conditions that favor the formation of base pairs between the probes and the target sequences. After washing away the excess sample, the microarray is scanned by a laser that excites the fluorescence of the bound targets. The intensity and color of the fluorescence indicate the presence and quantity of the target sequences in the sample.

By comparing the fluorescence signals from different samples, such as healthy and diseased tissues, researchers can identify genes that are differentially expressed, overexpressed, underexpressed, or silenced. They can also detect genetic variations that may be associated with certain phenotypes or diseases.

DNA microarrays have many applications in various fields of biology and medicine, such as genomics, transcriptomics, proteomics, pharmacogenomics, toxicogenomics, diagnostics, and drug discovery. They can provide insights into the molecular mechanisms of gene regulation, cellular function, development, differentiation, and disease. They can also help to discover new biomarkers, drug targets, and therapeutic agents.

However, DNA microarrays also have some limitations and challenges. They are expensive to produce and require specialized equipment and expertise to perform and analyze. They may produce large amounts of data that are complex and noisy. They may also suffer from technical errors, such as cross-hybridization, background noise, signal saturation, or dye bias. Therefore, they require careful design, quality control, normalization, and statistical methods to ensure reliable and reproducible results.

In this article, we will discuss the principle, types, requirements, steps, applications, advantages, and disadvantages of the DNA microarray technique in detail. We will also provide some examples of how DNA microarrays are used in different fields of research and practice.

The principle of DNA microarrays lies in the hybridization between the nucleic acid strands. Hybridization is the process of forming a double-stranded molecule from two complementary single-stranded molecules. The complementary nucleic acid sequences are able to specifically pair with each other by forming hydrogen bonds between the matching nucleotide base pairs. For example, adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G) in DNA.

To perform a DNA microarray experiment, the samples of interest are labeled using fluorescent dyes. The samples can be DNA or RNA molecules derived from different sources, such as healthy and diseased cells or different tissues or organisms. The labeled samples are then hybridized into the chip, which contains thousands of probes attached to a solid surface. The probes are short DNA sequences that are designed to match specific regions of the samples. Each probe corresponds to a gene or a part of a gene that is being studied.

When the labeled samples and the probes come into contact, they will bind to each other if they have complementary sequences. This is called specific binding or specific hybridization. Non-specific binding or non-specific hybridization occurs when the samples and the probes bind to each other without having complementary sequences. This can happen due to random interactions or cross-hybridization. The non-specific binding sequences are removed by washing the chip after the hybridization step.

The hybridized chip is then scanned using a device that can detect the fluorescent signals emitted by the labeled samples. The intensity and the color of the signals depend on the amount and the type of samples that are bound to each probe. The signals are captured by a camera and stored in a computer for further analysis. The data obtained from the scanning can reveal information about the gene expression levels, gene variations, gene interactions, and gene functions of the samples.

Using this technology, the presence and the quantity of one genomic or cDNA sequence in 100,000 or more sequences can be screened in a single hybridization. This allows researchers to study the global patterns of gene activity and regulation in various biological systems.

DNA microarrays can be classified into two main types based on the nature and length of the DNA probes that are attached to the solid surface. These are:

• Spotted DNA arrays or cDNA arrays: These are microarrays that use cDNA (complementary DNA) as probes. cDNA is synthesized from mRNA (messenger RNA) by reverse transcription, and it represents the expressed genes in a given sample. cDNA probes are usually longer than 100 base pairs, and they can be generated from any source of mRNA, such as tissues, cells, or organisms. cDNA probes are spotted on the solid surface using a robotic device or a pin. Each spot can contain thousands of copies of the same cDNA probe. Spotted DNA arrays can be used to compare gene expression levels between two or more samples by hybridizing them with differentially labeled cDNA targets.

• Oligonucleotide arrays or Gene Chips: These are microarrays that use synthetic oligonucleotides (short DNA sequences) as probes. Oligonucleotide probes are usually 20 to 25 base pairs long, and they are designed to match specific regions of the genome or transcriptome. Oligonucleotide probes are synthesized directly on the solid surface using a photolithographic process, which allows for high-density and high-precision arrays. Each spot can contain millions of copies of the same oligonucleotide probe. Oligonucleotide arrays can be used to detect single nucleotide polymorphisms (SNPs), gene copy number variations, alternative splicing events, or gene expression levels by hybridizing them with labeled DNA or RNA targets.

Both types of DNA microarrays have their advantages and disadvantages depending on the research question and the available resources. Some of the factors that can influence the choice of microarray type are:

• Coverage: Spotted DNA arrays can cover a wider range of genes than oligonucleotide arrays, especially for non-model organisms or novel transcripts. However, oligonucleotide arrays can provide more comprehensive and accurate information about each gene, such as its expression level, sequence variation, or splicing pattern.

• Specificity: Spotted DNA arrays can suffer from cross-hybridization or non-specific binding between probes and targets, which can reduce the signal-to-noise ratio and affect the data quality. Oligonucleotide arrays have higher specificity and sensitivity due to their shorter and more precise probes, which can discriminate between closely related sequences.

• Cost: Spotted DNA arrays are generally cheaper and easier to produce than oligonucleotide arrays, as they do not require sophisticated equipment or chemical synthesis. However, oligonucleotide arrays have lower operating costs and higher reproducibility than spotted DNA arrays, as they do not require labeling or amplification of the target samples.

• Flexibility: Spotted DNA arrays offer more flexibility and customization than oligonucleotide arrays, as they can be designed and printed according to the researcher`s needs and interests. However, oligonucleotide arrays have more standardization and compatibility than spotted DNA arrays, as they are produced by commercial companies and follow common formats and protocols.

In summary, DNA microarrays can be divided into two types: spotted DNA arrays and oligonucleotide arrays. Each type has its own strengths and limitations depending on the research objective and the available resources. Therefore, it is important to choose the appropriate type of microarray for each experiment.

Spotted DNA arrays ("cDNA arrays")

Spotted DNA arrays are a type of DNA microarrays that use cDNA (complementary DNA) as probes. cDNA is synthesized from mRNA (messenger RNA) by reverse transcription, and it represents the genes that are expressed in a given sample. Spotted DNA arrays are also called cDNA arrays or probe DNA.

To prepare spotted DNA arrays, cDNAs are amplified by using PCR (polymerase chain reaction), a technique that makes multiple copies of a DNA segment. Then, these cDNAs are immobilized on a solid support made of a nylon filter or glass slide (1 x 3 inches). The cDNAs are loaded into a spotting pin by capillary action, and a small volume of this DNA preparation is spotted on the solid surface by making physical contact between them. The DNA is delivered mechanically or in a robotic manner.

Spotted DNA arrays can be used to compare gene expression levels between two or more samples. For example, one can compare the gene expression profiles of healthy cells and diseased cells or of cells treated with different drugs. To do this, the mRNA from each sample is isolated and labeled with different fluorescent dyes. Then, the labeled cDNAs from each sample are mixed and hybridized to the spotted DNA array. The hybridization occurs between the complementary sequences of the cDNAs and the probes on the array. The non-specific binding sequences are washed out during the washing step of the process.

The hybridized array is then scanned by a laser that excites the fluorescence of the cDNAs, generating signals that depend on the amount and type of cDNA bound to each spot. The signals are captured by a camera and analyzed by a computer. The difference in the intensity and color of the signals for each spot indicates the relative expression level of the corresponding gene in each sample.

Spotted DNA arrays have some advantages over other types of DNA microarrays, such as:

• They are cheaper and easier to produce, as they do not require sophisticated equipment or techniques.

• They can be customized to include any genes of interest, as they do not depend on predefined sets of probes.

• They can detect alternative splicing events, as they use longer cDNA sequences that cover more regions of the genes.

However, spotted DNA arrays also have some limitations, such as:

• They have lower resolution and accuracy, as they use larger spots and fewer probes per gene.

• They have higher variability and noise, as they depend on manual or robotic spotting and washing steps.

• They have lower reproducibility and comparability, as they may differ in quality and content from batch to batch or from lab to lab.

Oligonucleotide arrays are another type of DNA microarray that uses short DNA sequences (oligonucleotides) as probes. Unlike cDNA arrays, oligonucleotide arrays are not prepared from PCR-amplified DNA fragments but are synthesized directly on the solid support using chemical methods. The most common technique for synthesizing oligonucleotide arrays is photolithography, which is borrowed from the computer industry. Photolithography uses light and masks to control the addition of nucleotides to specific locations on the array.

Oligonucleotide arrays have some advantages over cDNA arrays. First, they can be designed to target specific regions of the genes, such as exons, introns, splice variants, or SNPs. This allows for a more precise and comprehensive analysis of gene expression and function. Second, they can be standardized and mass-produced, which reduces the variability and cost of the arrays. Third, they can have higher density and resolution, which enables the detection of more genes and subtle changes in expression levels.

One of the most widely used oligonucleotide arrays is the GeneChip, developed by Affymetrix. A GeneChip typically contains millions of oligonucleotide probes that are 25 nucleotides long. Each gene is represented by multiple probes that cover different regions of the gene. The probes are grouped into probe sets, which are used to measure the expression level of a gene or a transcript variant. The GeneChip can also be customized to include probes for specific genes or applications.

To use a GeneChip, the target sample (usually mRNA or cDNA) is first labeled with a fluorescent dye and then hybridized to the array. After washing away the excess sample, a scanner is used to measure the fluorescence intensity of each probe on the array. The intensity reflects the amount of hybridization between the probe and the target sample. The data are then analyzed using software tools that compare the intensity of different probe sets and calculate the expression levels of the genes or transcripts.

Oligonucleotide arrays have been widely used for various applications in genomics, such as gene expression profiling, alternative splicing detection, SNP genotyping, copy number variation analysis, and comparative genomic hybridization. They have also been applied to study various biological processes and diseases, such as development, differentiation, cell cycle, apoptosis, cancer, infection, inflammation, and drug response.

There are certain requirements for designing a DNA microarray system, such as:

• DNA Chip: This is the solid surface where the probes are attached. It can be made of glass, silicon, nylon, or other materials. The chip can have thousands of spots, each containing a specific DNA sequence that corresponds to a gene or a part of a gene. The chip can be either spotted or synthesized on the surface.

• Target sample: This is the source of DNA or RNA that we want to analyze using the microarray. The target sample can be extracted from cells, tissues, blood, or other biological materials. The target sample can be labeled with fluorescent dyes or other markers to make it visible on the chip.

• Fluorescent dyes: These are molecules that emit light of a specific wavelength when excited by a laser. Different dyes can have different colors, such as green, red, blue, or yellow. Fluorescent dyes are used to label the target sample so that it can be detected on the chip.

• Probes: These are short DNA sequences that are complementary to the target sample. Probes are attached to the chip and hybridize with the target sample when they are exposed to each other. Probes can be either cDNA (complementary DNA) or oligonucleotides (short synthetic DNA).

• Scanner: This is a device that scans the chip and measures the fluorescence intensity of each spot. The scanner consists of a laser, a camera, and a computer. The laser excites the fluorescent dyes on the chip, and the camera captures the images. The computer stores and analyzes the data and generates a report.

These are some of the basic requirements for performing a DNA microarray experiment. Depending on the type and purpose of the experiment, there may be additional steps or components involved. For example, some experiments may require amplification, normalization, quality control, or statistical analysis of the data.

The reaction procedure of DNA microarray takes place in several steps:

1. Collection of samples: The sample may be a cell/tissue of the organism that we wish to conduct the study on. Two types of samples are collected: healthy cells and infected cells, for comparison and to obtain the results.

1. Isolation of mRNA: RNA is extracted from the sample using a column or solvent like phenol-chloroform. From the extracted RNA, mRNA is separated, leaving behind rRNA and tRNA. As mRNA has a poly-A tail, column beads with poly-T-tails are used to bind mRNA. After the extraction, the column is rinsed with buffer to isolate mRNA from the beads.

1. Creation of labeled cDNA: To create cDNA (complementary DNA strand), reverse transcription of the mRNA is done. Both the samples are then incorporated with different fluorescent dyes for producing fluorescent cDNA strands. This helps in distinguishing the sample category of the cDNAs.

1. Hybridization: The labeled cDNAs from both samples are placed in the DNA microarray so that each cDNA gets hybridized to its complementary strand; they are also thoroughly washed to remove unbounded sequences.

1. Collection and analysis: The collection of data is done by using a microarray scanner. This scanner consists of a laser, a computer, and a camera. The laser excites the fluorescence of the cDNA, generating signals. When the laser scans the array, the camera records the images produced. Then the computer stores the data and provides the results immediately. The data thus produced are then analyzed. The difference in the intensity of the colors for each spot determines the character of the gene in that particular spot.

DNA microarray is a powerful tool that can be used for various purposes in biology and medicine. Some of the applications of DNA microarray are:

• Detection of single nucleotide polymorphisms (SNPs): SNPs are variations in a single base pair of DNA that occur in the population. They can be used to identify genetic markers for diseases, traits, or ancestry. DNA microarray can be used to screen thousands of SNPs in a single experiment and compare them between different individuals or groups.

• Genetic mapping: Genetic mapping is the process of locating genes on chromosomes based on their linkage to other genes or markers. DNA microarray can be used to generate high-resolution maps of genomes by measuring the hybridization patterns of DNA fragments from different sources.

• Proteomics: Proteomics is the study of the structure, function, and interactions of proteins in a cell or organism. DNA microarray can be used to measure the expression levels of thousands of genes simultaneously and infer the activity and regulation of their corresponding proteins.

• Cancer diagnosis and prognosis: Cancer is a disease characterized by abnormal and uncontrolled growth of cells. DNA microarray can be used to identify the genes that are altered or mutated in cancer cells and compare them with normal cells. This can help to diagnose the type and stage of cancer, predict the response to treatment, and monitor the progression of the disease.

• Infectious disease diagnosis and surveillance: Infectious diseases are caused by microorganisms such as bacteria, viruses, fungi, or parasites. DNA microarray can be used to detect and identify the pathogens that cause infectious diseases by analyzing their genomic or transcriptomic profiles. This can help to diagnose the infection, determine its source and transmission, and monitor its spread and evolution.

• Drug discovery and development: Drug discovery and development is the process of finding and testing new compounds that can modulate biological targets for therapeutic purposes. DNA microarray can be used to screen potential drug candidates for their effects on gene expression and identify their mechanisms of action, efficacy, and toxicity.

• Pharmacogenomics: Pharmacogenomics is the study of how genetic variations affect the response to drugs. DNA microarray can be used to analyze the genetic factors that influence the metabolism, transport, and action of drugs in individuals or populations. This can help to optimize drug dosage, efficacy, and safety based on personal or group characteristics.

These are some of the applications of DNA microarray that demonstrate its versatility and utility in various fields of research and practice. DNA microarray technology has revolutionized our understanding of biology and medicine by providing a comprehensive and high-throughput approach to analyzing nucleic acids at a genome-wide scale.

DNA microarray is a powerful technique that allows researchers to study the expression of thousands of genes simultaneously. It has many advantages over traditional methods of gene analysis, such as:

• It provides data for thousands of genes in real-time. This enables researchers to monitor the changes in gene expression under different conditions, such as disease, stress, drug treatment, etc. It also allows them to compare the expression profiles of different samples, such as normal and diseased tissues or different individuals or species.

• It generates many results easily with a single experiment. This reduces the time and cost of performing multiple experiments with different methods. It also increases the efficiency and accuracy of data collection and analysis.

• It is fast and easy to obtain results. The process of hybridization, scanning, and data analysis can be automated and completed within hours or days. The results can be visualized and interpreted using software tools and databases.

• It is promising for discovering cures for diseases and cancer. By identifying the genes that are involved in various biological processes and pathways, researchers can understand the molecular mechanisms of diseases and develop new diagnostic tools and therapeutic strategies. For example, DNA microarray can help identify biomarkers for early detection of diseases or drug targets for personalized medicine.

• It can use different parts of DNA to study gene expression. Depending on the type of DNA microarray, researchers can use cDNA, oligonucleotides, or genomic DNA as probes to hybridize with the target samples. This allows them to study different aspects of gene expression, such as transcription, splicing, mutation, etc.

Despite the many advantages of DNA microarray technology, there are also some limitations and challenges that need to be addressed. Some of the disadvantages of DNA microarray are:

• High cost: DNA microarray experiments require specialized equipment, materials, and expertise, which makes them expensive to perform. The cost of a single DNA chip can range from hundreds to thousands of dollars, depending on the number and type of probes. Moreover, the analysis and interpretation of the data generated by DNA microarray can also be costly and time-consuming, requiring sophisticated software and computational resources.

• Complexity: DNA microarray produces a large amount of data that can be difficult to manage, process, and interpret. The quality and reliability of the data depend on many factors, such as the design of the probes, the hybridization conditions, the labeling and detection methods, the normalization and statistical methods, and the biological relevance and reproducibility of the results. Moreover, the data from different platforms or experiments may not be comparable or compatible due to variations in protocols and standards.

• Noise and bias: DNA microarray data can be affected by various sources of noise and bias that can introduce errors or artifacts in the measurements. For example, the background signal from non-specific hybridization or cross-hybridization can interfere with the detection of low-abundance transcripts or subtle changes in gene expression. The choice of reference sample or control group can also influence the outcome of the analysis. Furthermore, the selection of probes or genes to be included in the array can introduce bias or miss important information that is not represented on the chip.

• Limited scope: DNA microarray technology mainly focuses on measuring the abundance or expression level of mRNA transcripts in a given sample. However, this does not necessarily reflect the actual activity or function of the corresponding proteins or genes in the cell. There are many post-transcriptional and post-translational modifications and interactions that can affect the regulation and function of genes and proteins that are not captured by DNA microarray. Moreover, DNA microarray cannot detect novel or unknown genes or transcripts that are not present on the chip.

These are some of the disadvantages of DNA microarray technology that limit its applicability and accuracy in some cases. However, with continuous improvement and innovation in this field, these challenges can be overcome or minimized in the future. DNA microarray remains a powerful and versatile tool for studying gene expression and function in various biological systems.

We are Compiling this Section. Thanks for your understanding.