What Is Array Comparative Genomic Hybridization?

Introduction

Most genetic disorders are caused by unbalanced chromosomal abnormalities, which result in net gain or loss of genetic material. Cytologists have traditionally detected such abnormalities by creating a karyotype of a person’s chromosomes and analyzing the banding patterns contained within. However, researchers have increasingly relied on newer cytogenetic techniques in recent years. Array comparative genomic hybridization is a new molecular technique implemented to overcome the limitations associated with traditional cytogenetic techniques.

What Is Comparative Genomic Hybridization?

Comparative genomic hybridization (CGH) is a popular technique for screening for copy number variations across the genome. CGH employs two genomes, one test, and one control, differentially labeled and competitively hybridized to metaphase chromosomes. The fluorescent signal intensity of the labeled test DNA (deoxyribonucleic acid) compared to the reference DNA can then be plotted linearly across each chromosome, allowing copy number changes to be identified. The main objective of CGH is to quickly and effectively compare two genomic DNA samples obtained from two sources, which are frequently connected because it is possible that they have different chromosome or subchromosomal gains or losses.

What Is Array Comparative Genomic Hybridization?

The array comparative genomic hybridization (aCGH) technique detects chromosomal copy number changes on a genome-wide and high-resolution scale. Array comparative genomic hybridization (aCGH) overcomes the limitations of conventional cytogenetic analysis. The principles of CGH have been combined with the use of microarrays in this technique. It uses arrays of slides containing small DNA segments as the analysis targets rather than metaphase chromosomes.

Array CGH uses the same competitive fluorescence in situ hybridization principles as traditional CGH to compare the patient’s genome to a reference genome, identify the differences between the two genomes, and locate regions of genomic imbalances in the patient. With significant advantages over other techniques used for analyzing DNA copy number changes, array CGH has shown to be a precise, sensitive, quick, and high-throughput technique, making it more suitable for diagnostic applications. This technique allows for detecting DNA sequence copy number changes at a level of 5 to 10 kilobases.

What Are the Steps Involved in Array Comparative Genomic Hybridization?

Array CGH works on the same principles as traditional CGH. DNA from a control or reference sample and DNA from a patient or test sample are differentially labeled with fluorophores and used as probes that hybridize competitively onto nucleic acid targets in both techniques. The target in conventional CGH is a reference metaphase spread. These targets in array CGH can be genomic fragments cloned in various vectors such as bacterial artificial chromosomes (BACs) or plasmids, cDNAs (complementary DNA), or oligonucleotides. The steps involved in array comparative genomic hybridization are the following.

  • The test sample DNA is labeled with a red fluorophore, while the reference DNA sample is labeled with a green fluorophore.

  • Equal amounts of the two DNA samples are mixed and hybridized into a DNA microarray of several thousand evenly spaced cloned DNA fragments, or oligonucleotides, spotted in triplicate on the array.

  • Digital imaging systems are used after hybridization to capture and quantify the fluorescence intensities of each hybridized fluorophore.

  • If the fluorochrome intensities on one probe are equal, this region of the patient’s genome is interpreted as an equal amount of DNA in the test and reference samples; if the ratio is altered, this indicates a loss or gain of the patient’s DNA at that specific genomic region.

What Are the Applications of Array Comparative Genomic Hybridization?

The use of array CGH in research has accelerated the rate of gene discovery in human genetics, deepened the understanding of genomic alterations in cancer, and advanced the study of fundamental ideas related to chromosome conformation, DNA methylation, gene silencing, histone acetylation, replication timing, and other fundamental mechanisms referring to DNA structure and function.

  • Genomic Abnormalities in Cancer – Genetic changes and rearrangements are common in cancer and contribute to its pathogenesis. The detection of these aberrations by an array of comparative genomic hybridization provides information on the locations of cancer genes, which can be used in clinical diagnosis, cancer classification, and prognosis.

  • Prenatal Genetic Diagnosis – Preimplantation genetic screening using array CGH is a concept that is gaining popularity. It may be able to identify copy number variations (CNVs) and aneuploidy (abnormal number of chromosomes) in sperm, eggs, or embryos that could cause miscarriage, failure to successfully implant the embryo or other problems like Down syndrome (trisomy 21). This makes array CGH a potentially useful tool for lowering the prevalence of life-altering conditions and raising IVF (in vitro fertilization) success rates.

What Are the Advantages of Array Comparative Genomic Hybridization?

  • The main benefit of array comparative genomic hybridization (aCGH) is the simultaneous detection of deletions, aneuploidies, duplications, or amplifications at any locus represented on an array.

  • One assay using this technique is equivalent to thousands of FISH (fluorescence in situ hybridization) experiments, saving time and money.

  • Furthermore, aCGH has been demonstrated to be a potent tool for detecting submicroscopic chromosomal abnormalities in people with idiopathic mental retardation and various congenital disabilities.

  • Array comparative genomic hybridization has been shown in numerous large-scale studies to have a 10 to 20 % detection rate of chromosomal abnormalities in children with mental retardation or developmental delay. Only 3 to 5 % of these abnormalities would be detectable by other methods.

What Are the Limitations of Array Comparative Genomic Hybridization?

The major limitation of array CGH is its limited ability to detect mosaicism and inability to detect aberrations that do not lead to copy number changes. Such as triploidy, balanced reciprocal chromosomal translocations, transpositions, and inversions. The sensitivity and spatial resolution of the clones determine the degree of mosaicism that can be detected. Currently, the detection threshold is set at rearrangements that are present in roughly 50 % of the cells.

Conclusion

Array comparative genomic hybridization is a newer method of detecting copy number variations in DNA. In this technique, the whole genome is scanned for DNA copy number variations. Chromosomal imbalances of at least 5 Mb can be found using standard chromosome analysis. However, deletions and duplications that are not visible using standard chromosome analysis can be found using array comparative genomic hybridization.

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