DNA hybridization: techniques for chromosomal tests
Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes to detect deletions and duplications in DNA sequences or chromosomes. The term ‘cytogenetics’ is used to describe the branch of Genetics that studies the structure and function of the human cell, especially the chromosomes. It was developed in the early 1980s by biomedical researchers to refine specific features of DNA for use in genetic counseling, medicine, and species identification. FISH is also used to detect specific RNA (ribonucleic acid) targets in human cells, tissue samples, and tumor cells.
To understand fluorescence in situ hybridization, we need to understand a bit about DNA and chromosomes.
Chromosomes are structures within the cells of the human body that contain the DNA (deoxyribonucleic acid) or genetic coding that tells the body its development and functions. The human body has 46 chromosomes, arranged in 23 pairs; each pair has one chromosome from each parent arranged sequentially from number 1 to 22. The last pair of chromosomes determines gender: XX for girls and XY for boys. Cytogenetic or chromosomal testing is done when it is necessary to identify changes in the number and structure of genetic material and look for gains (duplications) and losses (deletions) to understand birth defects, developmental abilities, and disabilities in humans. .
Scientists and clinical researchers view and study chromosomes under a microscope through a process of staining and magnification. This helps them look for changes by studying the banding patterns and shapes of chromosomes; if there are large changes, imbalances, and rearrangements (also known as duplications and deletions) involving part or all of the chromosome, these can be seen. However, even small gains and losses of genetic material are of great importance, and sometimes even the most experienced and trained clinical scientists can miss these small changes through routine chromosome testing.
This is where fluorescence in situ hybridization (FISH) comes into play. It uses a chemical that glows (fluorescence) to detect the specific area of a chromosome that needs to be studied. Using a special microscope, a scientist can identify if there are duplications and deletions of DNA material. The technique uses fluorescently labeled probes attached complementary to specific parts of DNA strands, which when heated ‘denature’ or break, allowing the probes to ‘hybridize’ to their complementary DNA sequence. In simple terms, the probe will bind (hybridize) to the sample if there are any genetic mutations present in the DNA material; if there isn’t one, it won’t.
So now we understand that with ‘hybridization’, we can detect the absence of the presence of specific genetic mutations on the chromosomes that could help detect a disability, illness or disease.
Cell line authentication
A ‘cell line’ is used for biological research and experimentation. These are products of ‘immortal cells’ such as cancer cells that can perpetuate indefinite division, as opposed to regular cells that can divide up to about 50 times. These ‘immortal cells’ are extremely useful because they are readily available as a research product in laboratories and do not require ‘cell harvesting’, which is acquiring tissue from a human donor each time a laboratory needs cells for research.
Cell lines are the most important factors to study tumor biology, especially under laboratory conditions. However, the risks of cross-contamination and misidentification of cell lines in laboratories are quite high. Especially in investigations where the integrity of the results is paramount, reliable procedures for the authentication and identification of cell lines should be the main measures for quality control. By using multiplex fluorescence in situ hybridization and comparative genomic hybridization, genetic similarities between cultured cell lines and corresponding tumor tissues can be revealed.
Extensive and well-documented reviews of preventive measures and cases show that the three most useful methods for authentication and identification of cell lines are:
1. Karyology – basic karyotyping, special karyotyping, marker chromosomes, G and C chromosomal banding, and fluorescent in situ hybridization.
2. Isoenzymes: extraction of proteins and isoenzymes, separation and identification of patterns and application to the identification between species.
3. DNA Fingerprinting: DNA profiling and base sequences, use of automated sequencing, and detection of fluorescent labels.
