DNA Microarray- Definition, Principle, Procedure, Types, Uses

DNA microarray is a technology that is used to measure the relative concentration of nucleic acid sequences in a mixture where thousands of nucleic acids are bound to the surface through a hybridization process.

Microarray is a technology that uses a solid surface called a chip that involves binding thousands or millions of nucleic acid fragments onto the same solid surface.

Introduction of DNA microarray

  • It is also called the DNA chip, DNA array, gene chip, and biochips.
  • The latest generation of biosensors are biochips that are developed by the use of DNA probes.
  • It is the collection of the DNA on the solid surface known as the chip.
  • DNA microarrays are usually made up of silicon or glass to which the DNA is attached.
  • The expression of tens and thousands of genes can be examined with the help of a DNA microarray.
  • cDNA-based microarray and oligonucleotide-based microarray are the two different types of microarray.
  • For high-throughput and large-scale genomic analysis, the most successful and mature form of methodologies is used, known as DNA microarray.
  • Initially, this technology was developed to measure the transcriptional level of RNA transcripts extracted from thousands of genes.
  • DNA microarray research is not just limited to gene expression but is used to detect single nucleotide polymorphism (SNPs), methylated patterns, alternative RNA splicing, alteration in the gene copy number, and pathogen detection.
DNA microarray
DNA microarray

Principle of DNA microarray

  • The main principle of DNA microarray is based on the hybridization method, where the complementary nucleotide specifically pairs with each other by the formation of hydrogen bonds between base pairs.
  • When the number of the complementary base pairs nucleotide is higher, the number of non-covalent bonds between the base pairs is tighter.
  • The tightly paired strands remain hybridized even after washing off the molecules.
  • Generation of the signals occurs when the fluorescence-labeled targeted sequence binds to the probe, and the generated signal strength depends upon the amount of the target sequence bound to the specific probes.
  • In different conditions, the relative intensity of the spot is compared with the intensity of another spot.
  • During hybridization, a DNA duplex is formed when the sequence complementary to the probe will anneal, and the DNA target diffuses passively across the glass surface.
  • The detection of hybridized targets can be performed by one of many reporter molecule systems.
  • DNA microarray is a reverse dot blot that was used for many years with membrane-bound nucleic acids called southern and Northern bolts.
  • The qualitative and quantitative properties of the chip can be examined by the use of autoradiography, laser scanning, fluorescent detection device, and enzyme detection systems after the hybridization has been completed.

DNA microarray Procedure

It includes the following steps:

  1. Sample collection

Samples can be collected from a wide range of organisms. For example, it can be cancerous human skin tissue and healthy human skin tissue which can be compared and the analysis can be done. Samples can be tissue or cells that we want to work on.

  1. mRNA isolation
  • RNA is extracted from the sample by the use of the column or phenol-chloroform solvent.
  • Then mRNA is isolated, leaving behind rRNA and tRNA after the extraction of the RNA from the sample. We can use the column containing beads with poly-T tails to bind mRNA as mRNA has poly-A tails.
  • After this, mRNA is released from the beads by rinsing the solution with the buffer. The buffer has the ability to disturb the pH leading to alterations in the hybrid bonds.
  1. Labeled cDNA creation
  • The RNA is added to the labeled mix and this labeled mix contains poly-T (oligo dT) primers, reverse transcriptase, and fluorescently dyed nucleotides.
  • Reverse transcriptase is used to make cDNA.
  • After this cyanine 3 (cy3-fluoresces green) and cyanine 5 (cy5-fluoresces red) is added to the healthy cells and cancerous cells respectively.
  • At first, the primer and RT bind to the mRNA and then the fluorescently dyed nucleotide, forming a complementary strand of DNA.
DNA microarray procedure
DNA microarray procedure
  1. Hybridization 
  • From both the (healthy and cancerous) samples, the labeled cDNA that is prepared is placed on the DNA microarray which permits the hybridization of each cDNA to its complementary strand.
  • After this, the unpaired sequences are removed by a thorough wash.
  1. Detection of the intensities and analyzing the collected data
  • Relative intensities of fluorescence are detected by the use of a microarray scanner.
  • The scanner is equipped with a laser, camera, and computer.
  • The hybrid bonds that are present in the sequences are fluorescence with the help of the laser.
  • When the laser scans the plate, the camera records the produced images.
  • The computer stores our data and helps us to visualize our results immediately.

Types of DNA Microarray

  • cDNA-based microarray
  • oligonucleotide-based microarray

cDNA-based microarray

  • cDNA microarray is also called a spotted microarray, which is one of the first widely and broadly available array platforms.
  • For the preparation of chips, cDNA is used.
  • PCR is used for the amplification of the cDNA.
  • cDNA-based microarray is a high throughput technique.
  • This microarray technique allows the quantitative analysis of the transcribed RNAs from both known and unknown genes and is also called a highly parallel RNA expression assay technique.
  • Glass cDNA microarray is relatively affordable and does not require specific equipment for hybridization, detection sensitivity is increased due to longer targeted sequences, and to print a DNA sequence, primary sequence information is not needed.

Oligonucleotide based microarray

  • Oligonucleotide microarray hybridizes with only a single sample and gives the absolute levels of expression of the samples as it is highly specific.
  • During this method, the photolithographic technique is used to generate high-density microarray chips as the array is constructed by synthesizing single-stranded oligonucleotides in situ.
  • The collection and storage of the cloned DNA and PCR product are not required during this method.
  • Gene expression and analysis are limited in this method as it requires a large number of biological materials.
  • It is highly specific, fast, and reproducible.
  • The dsDNA probes are easier to produce; despite this, oligonucleotides need to be designed carefully so that all probes acquire similar melting temperatures and eliminate palindromic sequences.
  • Due to the smaller size, the probe cross-link can result in significant loss because of washing.
  • The modified 5’ to 3’ ends on coated slides help in the coupling of probes to the microarray surface that finally gives the functional groups like epoxy or aldehyde.

Applications of DNA microarray

  • DNA microarray is used for the analysis of transcriptomes and proteomes.
  • Several pathogenic and genetic diseases are diagnosed with the help of gene chips. 
  • DNA microarray can be used for the identification of the microbes present in the environment with the help of species-specific probes.
  • Genome genotyping through single nucleotide polymorphisms (SNPs).
  • DNA microarray is used to analyze cDNA produced from mRNA and detect different types of cells at different times.
  • It helps to measure the changes in gene expression by comparing the gene expression of two different samples.
  • This technique is also used for observing the mutations in the DNA.
  • DNA sequencing and drug discovery.
  • Study of genomes that are functional and profiling of genes that are expressed.
  • Helps in the study of proteomics.
  • Toxicological research and genetic engineering.

Advantages of DNA microarray

  • They have well-defined protocols for hybridization.
  • They also have well-defined pipelines.
  • For the submission of the data, there are standard approaches.
  • It is highly specific and of low cost.

Limitations of DNA microarray

  • Predefined sequences are only analyzed.
  • Limitation of the dynamic range because of the scanner.
  • It relies on hybridization, and hybridization is potentially non-specific.
  • Paralogue information might not be acquired.
  • Variance is higher even for lower expressed genes.
  • Splice variants are not generally identified.

Microarray-based on the type of Probes

  • DNA microarray:

DNA microarray measures the DNA and is also called a gene chip, DNA chip, or biochip. DNA microarrays are of different types that include, cDNA microarray, oligo DNA microarray, BAC microarrays, and SNP microarray.

  • MMChips: 

MMChips allow the analysis of laboratory data and studies of the interaction between DNA and Proteins.

  • Protein microarrays:

This microarray technique helps to analyze hundreds and thousands of proteins parallelly. Similarly, protein microarray is of different types that include, analytical protein microarrays, functional protein microarrays, and reverse-phase protein microarray.

  • Peptides microarray:

Peptide microarray is used for the study of protein-protein interactions in detail and helps in recognition of antibodies by screening proteomes.

  • Tissue microarray:

Tissue microarray is mostly used in pathology.

  • Antibody microarray:

Antibody microarrays are protein-specific microarrays that are used for the detection of antigens. It is also known as antibody array or antibody chips.

  • Cellular microarrays:

Large-scale chemicals and genomic libraries are screened by the cellular microarray technique. It is also used for the investigation of cellular microenvironments and is known as living-cell microarrays.

  • Chemical compound microarray:

This microarray technique is used for drug screening and drug discovery. This microarray is able to detect small molecules and is mostly used in the pharmaceutical industry.

  • Phenotype microarray:

Phenotypic microarrays are used to measure thousands of cellular phenotypes quantitatively and are also used in the drug development process. similarly, it is used in functional genomics and toxicological testing.

  • Reverse phase protein microarray:

Reverse-phase protein microarray is used mostly in clinical trials related to cancers. They are also used in the pharmaceutical industry, and in certain conditions, they can also be used in the study of biomarkers.

References

  1. https://www.genome.gov/genetics-glossary/Microarray-Technology
  2. https://www.genome.gov/about-genomics/fact-sheets/DNA-Microarray-Technology
  3. https://www.news-medical.net/life-sciences/Types-of-Microarray.aspx
  4. Fesseha H, Tilahun H. Principles and applications of deoxyribonucleic acid microarray: A review. Pathol Lab Med Open J. 2020; 1(1): 54-62. doi: 10.17140/PLMOJ-1-109
  5. Marco Wiltgen & Gernot P. Tilz (2007) DNA microarray analysis: Principles and clinical impact, Hematology, 12:4, 271-287, DOI: 10.1080/10245330701283967 To link to this article: https://doi.org/10.1080/10245330701283967
  6. https://molmed.biomedcentral.com/articles/10.2119/2006-00107.Trevino#citeas
  7. DNA microarrays: Types, Applications, and their future https://doi.org/10.1002%2F0471142727.mb2201s101
  8. https://www.biologyexams4u.com/2014/05/dna-microarray-steps-procedure.html
  9. https://www.biologyexams4u.com/search/label/Applications%20of%20DNA%20Microarray
  10. https://www.biologyexams4u.com/search/label/basic%20biotechnology%20notes
  11. https://www.biologyexams4u.com/search/label/DNA%20Microarray
  12. https://www.biologyexams4u.com/search/label/DNA%20Microarray%20steps
  13. https://www.biologyexams4u.com/search/label/Molecular%20Biology%20Techniques
  14. https://www.researchgate.net/figure/Summary-of-advantages-and-disadvantages-between-microarray-and-RNA-seq-approaches-All_fig3_298723854

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