DNA Microarrays
Principles and Construction
DNA microarrays are powerful tools for high-throughput genetic analysis, enabling the simultaneous measurement of the expression levels of thousands of genes. Here are the key principles and construction methods:
- Hybridization: The core principle is based on the hybridization between two complementary DNA strands, forming hydrogen bonds between nucleotide base pairs. This specific pairing allows for the detection of target sequences[1][4][5].
- Probe Immobilization: Probes, which are either spotted cDNAs or oligonucleotides, are immobilized in an ordered two-dimensional pattern on substrates such as glass slides or nylon membranes. These probes are designed to be specific for particular genes, genetic variants, or intergenic regions[1][4][5].
Methods of Creating Microarrays
- Pin Deposition: This method involves using small pins to transfer drops of DNA solution onto a solid substrate, resulting in spot sizes ranging from 50–360 µm[5].
- Ink-jet Printing: A non-contact method that reduces contamination risks, achieving high spot densities of up to 2500 spots/cm²[5].
- Photolithographic Techniques: Used for high-resolution arrays, this method synthesizes oligonucleotides directly on the substrate using light exposure, allowing for precise control of DNA sequence positions[5].
Applications
- Gene Expression Analysis: DNA microarrays are commonly used to compare gene expression levels between different samples, such as healthy vs. diseased cells or different organisms. mRNA is extracted, reverse-transcribed into cDNA, labeled with fluorescent dyes, and hybridized to the microarray. The fluorescence is then scanned to detect which genes are expressed[1][4][5].
- Genotyping and Polymorphism Detection: Microarrays can identify single nucleotide polymorphisms (SNPs) and other genetic variations, which is useful in forensic analysis, measuring disease predisposition, and evaluating germline or somatic mutations[1][4].
- Pathogen Detection and Antibiotic Resistance: DNA microarrays can detect pathogens, antibiotic resistances, and gene mutations, guiding therapy and public health interventions[4].
Data Analysis
- Hybridization and Scanning: After hybridization, the microarray is washed to remove non-specific hybrids, and a laser excites the fluorescent dyes. The resulting image is scanned and processed using specialized software to quantify the signal intensities of each spot[1][5].
- Normalization and Statistical Analysis: Raw data is normalized to account for background noise and variations in signal intensity. Statistical tests are used to determine whether gene expression is statistically different between sample groups[5].
Protein Microarrays
Principles and Construction
Protein microarrays are designed to study protein-ligand interactions and are crucial for understanding protein functions and behaviors.
- Recombinant Proteins: Proteins are typically produced in bacteria (e.g., E. coli) and then immobilized on a solid substrate[5].
- Interrogation Techniques: Various techniques are used to detect protein binding to other proteins, antibodies, or ligands. These include fluorescence, chemiluminescence, and other labeling methods[5].
Applications
- Antibody Arrays: These arrays assess antibody selectivity and cross-reactivity, and can study binding to various proteins. This is useful in diagnostics and therapeutic development[5].
- Interaction Studies: Protein microarrays help identify previously unknown protein interactions, such as those involving yeast proteins or platelet proteins[5].
- Enzyme Activity: Enzyme arrays can assess enzyme activities toward different substrates, providing valuable information for drug discovery and development[5].
Personalized Medicine
Role of DNA and Protein Microarrays
- Genetic Predispositions: DNA microarrays can identify genetic predispositions to diseases, enabling early screening and preventative measures. This is particularly useful for hereditary conditions and cancer[5].
- Tailored Treatments: By analyzing biopsies from patients, healthcare providers can determine susceptibility to specific chemotherapy drugs, optimizing treatment outcomes based on individual genetic and proteomic profiles[5].
Point-of-Care Testing
- Self-Testing Devices: Devices like those for diabetes monitoring track vital health parameters remotely, allowing for real-time adjustments to treatment and potentially preventing complications[5].
- Multi-Parameter Sensors: The development of sensors that monitor various health parameters (e.g., cholesterol, early-stage cancer markers) further enhances personalized, preventative healthcare by providing tailored treatments that maximize efficiency and improve outcomes[5].
Comparison with High-Throughput Sequencing
While DNA microarrays are powerful tools, high-throughput sequencing (HTS) offers several advantages:
- Unbiased View: HTS provides an unbiased view of the transcriptome, detecting transcripts and isoforms with higher specificity and sensitivity compared to microarrays[3].
- Dynamic Range: HTS offers a wider dynamic range and can detect differential expression more accurately. It also does not require the construction of a probe library, reducing errors and increasing accuracy[3].
- Versatility: HTS can analyze the composition and diversity of microbial communities and provide information on the functions encoded by the genomes of these communities, making it a versatile tool for various research applications[2][3].
