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Current cytogenetic technologies had redefined the margin which separates the cytological and the molecular cytogenetics. Since the discovery of the human chromosome in 1956, many methods had been invented to probe into the mysterious regions of the chromosomes. With new and advance techniques emerging, cytogenetic methodologies had evolved from being a microscopic technique, into a microarray technology. (Smeets, 2004) Using these new techniques, chromosomal aberrations could be visualized more accurately and the architecture and the functionalities of the chromosomes can be more clearly understood. Most of the cytogenetic methodologies focused more on tumour and cancer studies. These techniques are able combine with each other to form a more powerful technique to use. With these in mind, the future remains bright in the field of cytogenetics. More sensitive and powerful techniques which could increase in the resolution of the chromosomes would be invented to overcome the limitations of the previous technologies, as mentioned in this review.
Cytogenetics - a study of chromosomal evolution, structure and functions, can be traced back to the 19th century. This marked the beginning of the research in the field of cytogenetics. The first pictorial illustration of human chromosome was published by Flemming in 1882, and in 1888, what was previously named the 'stained body' was renamed as 'chromosome' by Waldeyer. (Smeets, 2004)
Chromosome was later thought to lay the foundation of heredity. However, due to the limitations of technology and scientific equipments, the study for cytogenetics was tough. Culturing of mammalian cells from various samples and fixing on slides for scientific studies was not at all possible as the samples were not of optimal conditions. Due to these restraints, the yields collected from the samples often contradict with the reported. One particular study conducted by Von Winiwarter reported the difference in the number of chromosomes between the sexes. In 1923, based on a clinical study of meiotic chromosomes in various testis biopsies extracted from the imprisoned, emasculated males who were formerly convicts, Painter justified that the number of chromosomes should be 48. This number attained recognition after a few years and it was used by the scientists for three decades. (Smeets, 2004)
In early 1950s, new discoveries conducted eventually led to the realization of the correct number of chromosomes found in man. Despite the successes noted in cell culturing as it was easy to access to divisible cells, it was still difficult to obtain nicely spread chromosomes. It was later an accidental discovery by Hsu, who immersed the culture into hypotonic salt solution before fixing them into the slides, that resulted in a more nicely spread chromosome structure. Along with this discovery, he too found that with the addition of colchicines to the cultured cells, the mitotic spindles would be damaged and destroyed while in the metaphase, which would result in the unfinished mitosis stage, leading to the rise in the number of metaphases which were feasible for microscopic studies. (Smeets, 2004)
The start of cytogenetics began in 1955 in Lund, Sweden, where an important discovery was made by Tjio and Levan. Levan, who collaborated with Hsu previously, cultured embryonic lung cells. Using the latest technology, they were able to obtain nicely spread chromosomes on slides which they were able to confirm the correct number of chromosomes was 46 instead of 48. However, despite their findings, they remained careful in publishing their results. Later the same year, the conclusion drawn from their experiment was further reaffirmed by the experiment carried out by Ford and Hamerton from their human spermatocytes experiment. In the experiment, the chromosomes were arranged according to their size and location of the centromeres, into eight different groups, A to G and the sex chromosomes. This arrangement enabled easy counting and defects in the chromosomes could be easily detected. (Smeets, 2004)
In 1966, Steele and Breg concluded that cultured cells obtained from the amniotic fluid could be used to study and determine the mutation and number of chromosomes in prenatal chromosomal studies. The results obtained from their studies illustrated that in abortion, over 50% of the aborted fetus demonstrated chromosomal aberrations, which led to the tetraploidy or triploidy, trisomy that were present either in chromosomes 13, 18, 21 or most frequently in 16, and alsoS monosomy in chromosomes 17 or 18. Comparing this result with other chromosomal results obtained from other mammals, it showed that in human chromosomes, chances of getting errors during chromosomal replication was higher, during the meiosis stage. The underlying reason for this unusual phenomenon is still under debate. It was thought that this phenomenon could be due to the increase in age of the mother, where the unbalanced offspring incidence raised from 1 in 250 at the age of 36 to 1 in 10 at the age of 45. In contrast, abnormal chromosomes were seen in one in seven abortions in young women. With this study, it was concluded that the error rate in meiotic failures that were present in humans were relatively high, as compared with other mammals. (Smeets, 2004)
2. Chromosome banding (G-banding)
In the late 1960s, detailed chromosomal banding analyses were conducted by coupling flurochromes with alkylating agent. This method was able to provide a fluorescence colour to the chromosomes, to enable the scientists to study and visualize the structure and banding of the chromosomes. In this way, a complete human karyotype could be obtained. In 1970s, scientific studies were performed on 'solid' stained chromosome interfered with the clear identification of each chromosomes and most of structural defects could be identified. Caspersson was able to demonstrate the banding pattern using fluorescent staining technique which incorporated the use of quinacrine and fluorescent microscopy. This technique was known as the quinacrine banding. Despite this, quinacrine banding method was not satisfying as the fluorescent intensity was not optimal, making it not feasible method to be used on subjects suspected of chromosomal defects. Due to this drawback, quinacrine banding technique was replaced by Giemsa banding swiftly. (Smeets, 2004)
2.2 First chromosome discovered by G-banding
Giemsa banding, also known as the G-banding technique, was accepted universally. This method is conducted by adding the enzyme trypsin to the cultured cell before staining with Giemsa stain. The bands in the chromosomes were obtained when the chromosomes were digested by trypsin (Fig.1.) G-banding had brought forth the increase in the studies in investigating the function of the chromosomes as it was an easy technique to use and at any one time, this technique required only a small sample to give optimal results. G-banding method allowed better resolution in the visualisation, so that genetic defects like chromosome translocation, deletion, inversion, and repetition could be identified easily. G-banding technique was not only used exclusively for chromosomal aberrations, but it was used to on healthy individuals to test if they were a carrier of the defective gene. Despite this useful technique, it was only revealed in 1973, after 13 years of its discovery. Using this method, the first acquired chromosome known as the Philadelphia chromosome (Speicher & Carter, 2005), revealed that the genetic defect was not caused by the deletion in chromosome 22, but a genetic translocation between chromosome 9 and 22. However, though the introduction of this method, the results obtained was still limited, with an approximation count of 500 bands per haploid genome. This problem was overcame by Yunis, who developed the high resolution banding by synchronizing the cultured lymphocytes, which in turn contributed the remarkable increase in the total number of
cells detected in the pro-metaphase or the prophase stage in the cell cycle. G-banding technique separated the regular metaphase bands in longer chromosomes into many smaller bands, doubling the number of bands detected in the genome; thereby increasing the resolution. In this way, more precise studies could be conducted on the chromosomes and mutations could be easily detected. (Smeets, 2004)
(Picture from Smeets, 2004)
2.3 Clinical Application of G-banding
Using this technique, chromosomal disorders which resulted in the clinical manifestation of several diseases could be linked to the micro-deletion of certain chromosome components or contiguous gene. The three good example of the application of this technique were the Prader Willi and Angelman syndrome, which the proximal arm of chromosome 15 was deleted, Smith-Magenis and Miller-Dieker syndrome was caused by the deletion of multiple different short proximal arm located on chromosome 17. The last example was DiGeorge/Velo Cardio Facial (VCF) syndrome, which was caused by the deletion of chromosomal components located at the long arm of chromosome 22. (Smeets, 2004)
Despite the advantages of G-banding, there were still some problems pertaining to the application of the technique. It could not accurately display chromosomal defects although it could show the chromosome under high- resolutions. Patients with clinical manifestations of Prader Willi and Angelman syndrome, Smith-Magenis and Miller-Dieker syndrome and DiGeorge/Velo Cardio Facial (VCF) syndrome illustrated but there were no chromosomal aberrations illustrated under this technique. Hence, new techniques, with higher resolution, were required urgently in order to accurately and precisely pinpoint the chromosome defects of the three syndromes. This led to the birth of the new technique called 'fluorescence in situ hybridization' or commonly referred as FISH, a technique which superceded the Giemsa's Banding technique. (Smeets, 2004)
2.5 Other applications of G-banding
G-banding is primarily used to produce the karyogram of the chromosomes. In the field of cancer studies, G-banding was the first technique to be used, in order to identify the abnormal and uncontrolled cell growth. Its role in the laboratory studies, was to aid the prediction and analysis of the causes of tumour or cancerous cells. G-banding also provides the restorative assessment of the cancerous cases.
G-banding technique had been the principle technique used to obtain the metaphase chromosomes for pre- and postnatal diagnostic applications for more than 20 years.
3. Fluorescence in situ hybridization (FISH)
In 1969, before the development of FISH technique, there was an in situ hybridization method developed as a technique that replaced G-banding. This method, co-founded by Joe Gall and Mary Lou Pardue, who discovered that by using DNA-RNA hybridization, they could pinpoint the genes that actually encode the ribosomal RNA (Speicher & Carter, 2005). This technique was an observation of the complementary sequences, which possessed the ability to anneal or merge with each other in order to form more stabilize DNA structures. This predecessor of FISH did not make the samples glow, instead, it used probes that were labeled with radioisotopes before performing any further studies. However, although this method was considered far more useful than G-banding and replaced G-banding swiftly, it had a couple of drawbacks. The probe must be at an unstable stage before it could be coupled with the radioactive material, but this disrupted the constant specific activity of the radioactive isotope as it would decay over time. Although the radioactive isotope was thought to have high sensitivity, but the resolution generated by them were limited. Due to the isotopes being exposed under extensive hours before any signal could be captured on the radiography film, this often led to the results of the assay being held up. These drawbacks were coupled with the fact that the radioactive probes posed a significant health risk, and thus led to the invention of FISH. (Levsky & Singer, 2003)
3.2 Principles of FISH (Fig.3)
FISH used a revolutionary new approach to detect chromosomal aberrations compared to G-banding. Instead of combining flurochromes with alkylating agent or the use of non-fluorescent radioactive probes, it used fluorescent probes which had the ability to bind to certain parts of the chromosomes illustrating high degree nucleic acid similarities. Several cycle of washing was performed in order to remove the probe DNA. After the completion of this step, the slide was examined under fluorescent microscopy, which would then be able to detect the position of the fluorescent probes which were bound to the chromosome (Fig. 2). This method not only conquered the limitations of G-banding, it also countered the problems which were noted in its predecessor. FISH excelled in giving better resolution to the chromosome, allowing better and precise location of the fluorescent probes, improved in the speed of the assay and it was relatively safe to use, compared to the radioactive isotopes. (Levsky & Singer, 2003) Following the footsteps of the Human Genome Project, there was a significant increase in the number of available probes manufactured for the purpose of diagnostic studies. These probes, like the cosmids, PACs, BACs, and YACs (Smeets, 2004), were produced by replicating and mapping of the human chromosomal segments.
Fig.2. Mechanism of conventional FISH. Chromosomal DNA is presented on the slide as metaphase chromosomes while the labeled probe DNA is added onto the slide for hybridization. After several rounds of washing steps to remove probe DNA, the slides are studied in fluorescent microscopy after the chromosomal DNA is stained. Picture taken from Smeets, 2004.
Fig.3. Flowchart of FISH technique. Picture taken from Moter & Gobel, 2000.
3.3 FISH at work
FISH was used extensively to study and analyze for various uncontrolled growth of abnormal tissue in tumour cytogenetics. The most apparent applications of FISH were the studying of the translocation of the large cell lymphoma, stem cells assay from acute myeloid leukaemia (AML) for patients with clinical manifestation of trisomy 8 and for the analysis of abnormal chromosomes of tumour in adolescent. (Kanna & Alwi, 2009)
Most commonly, FISH technique was used for validating the chromosomal defects, given that the probe used for the test is known. It was also used as an additional test for the chromosomal aberrations, apart from the traditional banding techniques.
3.4 Benefits of FISH technique
FISH was first applied in 1980. Using fluorophore as a probe for specific DNA sequences, it was labeled directly at the 3' end on the RNA. The luminous probe was prepared using fluorophore-modified bases coupling with specific enzymes. This method of preparing for the luminous probe had been used extensively, harmonizing one colour each time. Bases which were modified by the amino-allyl, were used to bind to fluorophore, which resulted in an orderly display of the low noise probes when applied with fundamental chemistry methods. Low copy number of nucleic acid assay was thus prevented in this method. With the binding of the secondary reporters to the hybridized probes, it allowed signal amplification. One of the advantages of this technique was its ability to apprehend results of multiple targets concurrently, using different fluorescent dyes. FISH also allowed the non-dividing chromosomal analysis of the nucleus to be conducted. (Levsky & Singer, 2003)
Direct detection was achieved through the enhanced labeling of the single-stranded DNA (ssDNA) probes procedure, where sufficient amount of hybrid probes with fluorescent capabilities were introduced. Since then, there had been a wide variety of methods available for usage in the direct or indirect labeling. With FISH, even the slightest aberrations in the chromosomes could be
identified, if the designated area is labeled with hybridized probe. This method allowed the DNA clones to be arranged in accordance to their bands and breakpoints. (Levsky & Singer, 2003)
3.5 FISH at its limits
However, although FISH could be considered as the state of the art tool to be used to visualize the chromosomes during that period, but this method did had its flaws. It was particularly tedious and time comsuming (Kannan & Alwi, 2009) as the fusion of the probes was necessary before any microscopic studies could be performed. Automating the probing process was hard, thereby limiting the number of probes that can be used. Probes of larger size were required to cut into smaller sizes, often not exceeding more than 200 nucleotides before they could be used. (Kearney, 2006)
4. Multicolur-FISH (mFISH) vs Spectral Karyotyping
4.1 Successor of conventional FISH
In 1989, there was a significant breakthrough in the FISH method. Nederlof and his team of scientists invented the method, known as the 'Multiplex-FISH', the successor of the two-colour detection (1986) and three-colour detection (1989) techniques. (Levsky & Singer, 2003)
4.2 Painting the world of chromosomes
Instead of presenting the results in black and white fashion, this method was able to give the chromosomes 'colours' for better study of the chromosomal aberrations by combining the use of blue coloured amino methyl coumarin acetic acid (AMCA), red coloured tetramethylrhodamine isothiocyanate (TRITC) and green coloured fluorescein isothiocyanate (FITC) (Liehr et al., 2006) (Garini, et al, 1996). By utilizing different fluorophore permutations to label the chromosomes, this could give each chromosome different 'colours' when screened for the different fluorochromes under the fluorescence microscope. The first experiment of its kind, which took place in 1989, involved only three chromosomes, each of them were labeled with different fluorophores and studied concurrently, attained success in 'colouring' the chromosomes, but the probe set only came into light in 1996. Using this probe set, not only it could present the full human chromosomes in a different shade, it could present the chromosomes all in different colour from in each other when coupled with the whole chromosome painting probes (wcp). To date, this probe set is marketed under many other names, like the multiplex-FISH (M-FISH), Combined Binary Ratio labeling-FISH (COBRA-FISH) or the 24-coloured FISH. (Fig 4) (Liehr, et al., 2006)
Picture taken from Kearney, 2006
In the 24-coloured FISH technique, in order to 'colour' all the twenty-four chromosomes individually, the nucleic acid from each of the chromosomes would have to be extracted before subjecting them to amplification. After amplification, a mixture of five dyes with fluorescence capabilities, inclusive of the newly incorporated cyanine dye, was added before studying under fluorescence microscope and interpreting the results with the analyzing computer. mFISH was proven to be particularly powerful to detect the complex chromosome detects like the chromosome translocations and reordering of chromosomes. 24-coloured FISH technique was able to give precise quantitation and positioning of each fluorochromes and at the same time, removing the need of chromatic crosstalk.
4. 3 Contrasting Spectral Kayotyping & mFISH
Spectral Karyotyping (SKY) - which coloured the whole chromosome in one single colour, when described by Malik and Garini in 1996 (Macville, et al., 1997), was termed as the technique which collaborate the use of spectroscopy and imaging. SKY use the same approach as mFISH, but instead of using the fluorescence microscope to differentiate and filter for different fluorochromes, an interferometer was used in place of this. SKY analyzed the fluorochrome under one exposure by using triple-bandpass optical filter, which allowed the red, blue and green colours to pass through the filter concurrently while restricting the wavelengths that would possibly hinder the transmission of the colours. This filter also did, removed the ultra-violet and infra-red rays, thereby allowing only the required hue to be transmitted. (Macville, et al., 1997) (Garini, et al., 1996)
4.4 Clinical applications of mFISH and SKY
mFISH and SKY were highly sensitive towards the complicated chromosomal deficiencies and detection of the chromosomal translocations. Particularly the mFISH technique, it focus on the studies for solid tumours that were normally distinguished by complicated chromosomal anormalies, acute myeloid leukaemia and acute lymphoblastic leukaemia. (Kanna & Alwi, 2009)
4.5 Favors of mFISH & SKY
Both techniques were able to probe into areas of unbalanced translocations, complicated alteration of chromosomes, and identification of chromosomal markers by staining the chromosomes, which increased the diminutive chromosomes by two fold. mFISH and SKY were also used to map the chromosomal breakpoints. (Kanna & Alwi, 2009)
4.6 Limitations of both techniques
Despite SKY and mFISH shared similar functionalities with each other, unfortunately, they shared the disadvantages as well. Both techniques were unsophisticated towards the defects such as inversions, duplications, or deletions which occur within the chromosomes and chromosomal breakpoints detection. For mFISH, although it could give the full colour pictorial representation of the human chromosomes by coupling with wcp, this could only be applied to the arrested chromosomes during metaphase, not to the ones present within the nucleus during the interphase stage of the cell cycle. Both techniques were also relatively more expensive, and technologically more challenging to handle. (Tonnies, 2002) Furthermore, right from the beginning, it was obvious that both techniques were unable to resolve the congenital problem of the probe set - low resolution presentation of the chromosomes. Those specimens with special requirements, like if vast number of fluorochromes used, would have to be sent to specialized laboratories for analyzing.
Hence, the need of inventing a brand new technique to overcome these disadvantages
surfaced. The technique, known as the Comparative Genomic Hybridization, had the ability to scan the full genome for amplification of chromosomes and this technique was developed as the alternative to the FISH method.
5. Comparative Genomic Hybridization (CGH)
Comparative genomic hybridization, also known as the Chromosomal Microarray Analysis (CMA), was first defined in 1992 (Wang, 2002) that this technique could be able to locate DNA acquisition and deficiencies with only one screening of the entire genome. Through this one screening, it could pinpoint the exact location of the chromosomal amplification as well as the pinpoint the exact location of the deletion on the specific chromosome.
5.2 Uniquely CGH: The Approach
For CGH, two DNA samples were being extracted, one from the test samples, and the other from the normal referencing sample as the control before labeling them with different dyes. After this, hybrid probes were being added. These hybrid probes would target at the chromosomes at metaphase stage and fight for the available complementary hybridization sites. Hence, if an amplification in the test sample was detected, the test sample would light up in the dye assigned for amplification. However, if a deletion was detected, it would light up in the dye assigned. The fluorescent ratio would be compared between the test sample and the control using the digital imaging system and specialized software for analyzing. (Wang, 2002) Depending on the results obtained for the test sample, the acquisition or deficiency of the chromosomal regions were often indicated by the intensification or the reduction of the ratio against the control.
5.3 CGH examples
The invention of CGH technique had given new insights to cancer. This technique was used mostly to investigate and study the complicated chromosomal aberrations of solid tumours like childhood hyperdiploid acute lymphoblastic leukaemia (Haas., et al., 1998), thyroid cancer (Chen, et al., 1998)and oral cancer , and it could be used for identification of the cancer related genes. (Inazawa, et al., 2004) It could be used to analyze and define the molecular classification of the Saccharimyces Sensu Stricto complex. (Ingram, et al., 2004)
5.4 Advantages of CGH
The invention of CGH had been used primarily as a discovery technique. The convenience of this technique is that, even without any knowledge of the chromosomal defects of the test sample prior to the test, it could still be used. This technique had ceased the need to develop any proliferation materials, and its flexibility of the specimen used for the test, had brought itself as a powerful technique to use in cytogenetic field. (Tonnies, 2002) This technique had the ability to give information on the chromosomal instability on the chromosome locus. CGH could also be used on small amount of DNA samples, like those obtained from micro-dissection. Such samples would have to be amplified using unbiased Polymerase Chain Reaction (PCR) method. This method could be used to analyze single cell samples too.
5.5 Restricted Actions of CGH
Despite CGH's capability in the cytogenetic field, its capacity is restricted when it
was used to analyze specimens which did not have any genomic inequality, such as when the genome had an equal number of translocations as well as inversions. In such cases, CGH is unable to give the information on the changes that occurred in the whole genome number. This technique was also not able to shed light on the architectural alteration of the chromosomes that were involved in the acquisition and deficiency. (Speicher & Carter, 2005)
These limitations had led to the invention of array based CGH or matrix array CGH in the late 1990s. This successor of the conventional CGH, was akin to be conducting several thousand of FISH technique in one single time. (Kanna & Alwi, 2009) This technique was able to elude all the disadvantages that existed in the conventional CGH method. Compared to the conventional CGH method, array-based CGH method could able to give higher resolution results. This method could detect the architectural alteration of the chromosome at minute amount of DNA sequences. It could provide more accurate information on the architectural breakpoints, acquisition and deficiencies located on the chromosomes, and it greatly reduced the time taken to conduct the full experimental investigation as compared to other cytogenetic methodologies.
Array-based CGH could be used on investigating the relationship existed in multiple synchronous tumours. (Wa, et al., 2005), lung cancer (Zhu, et al., 2005), bladder tumours (Veltman, et al., 2003) and identification of the subtypes of glioblastoma (Nigro, et al, 2005)
6. Other technique to visualize chromosomes
6.1 Sister Chromatid Exchange (SCE)
Discovered by Barbara McClintock in 1938, SCE was able to give a pictorial presentation of the exchanging of the brightly and dimly lit fluorescent portions of the sister chromatids during the anaphase of mitosis. This was achieved by including bromodeoxyuridine (BrdU) into the duplicating cells for 2 cellular cycles. The significance of SCEs is still unknown, but in carcinogenic cases, it was observed that there was an increase in the frequency of SCE. Increment of SCE could also be observed in patients suffering from clinical ankylosing spondiylitis, smokers, women exposed to the biomass fuels and patients with clinical carcinoma of the cervix uteri. (Kanna & Alwi, 2009) This method could be used to investigate cancer-induced diseases, such as the Bloom syndrome and the xeroderma pigmentosum. (Wang, 2002)
7. Cancer genetics
Chromosomal aberrations and abnormality of the chromosomal number which led to the suppression of tumour suppressor gene, would often account for tumorigenesis or carcinogenesis. Fusion proteins resulted from balanced chromosomal translocations would possess cancerous effects. A translocation located between chromosomes 9 and 22, would result to the establishment of the Philadelphia gene, with which this gene accounted for 95% of the cause of chronic myelogenous leukaemia (CML). (Kanna & Alwi., 2009)
CML was treated with Gilvec, a tyrosine kinase inhibitor (TKI) medication which would block the development of the protein produced by CML. Tyrosine kinases belonged to a group of proteins that mediated the signaling for cellular development. Gilvec had the ability to disrupt the cancerous growth by disrupting the transmission of growth signals to the cancerous cell. The progress of Gilvec treatment was monitored routinely using the conventional cytogenetic methodologies. Using this treatment, it was reported that there was a significant drop in the genes that would trigger the Philadelphia chromosome. (Kanna & Alwi, 2009)
Hence, cytogenetic studies played a crucial in this aspect. Results obtained from clinical trials would have to be analyzed carefully and cautiously, with which it could be used to segregate the patients into the different types of therapy. (Kanna & Alwi, 2009)
8. Conclusions and future outlook
Modern technologies in cytogenetics had redefine clearness of the cytological and molecular cytogenetic analysis. It is now widely accepted to use more than one technique to investigate the chromosome for its functions and structural changes. Although current cytogenetic methodologies were suffice in investigating chromosomal aberration, but molecular techniques which would increase in the resolution of the chromosomes give better understanding of the architectural, chromosomal capacity and the progress of the chromosomes were explored constantly. Conventional cytogenetic methodologies were still regarded as the preferred techniques used to obtain the full overview of the human genome. (Kanna & Alwi, 2009) Conventional banding techniques could be coupled with M-FISH and other techniques, they were used for investigate the symptoms presented in adolescents. A robust combination from the start, the CGH coupled with mFISH, was able to probe into complex chromosomes for more detailed analyzing.
Lately, microarray-based techniques had use cDNAs or oligonucleotides in place of the metaphase chromosomes as the targeted DNA for tests. (Kanna & Alwi, 2009) (Ylstra, et al., 2005) This heightened the resolution of the results significantly, and it could reflect the change in copy number directly to the genomic sequence. This illustrated the close knitted relationship between the chromosomal defects and the clinical manifestation of chromosomal disorders which existed in humans. Therefore, cytogenetic methodologies would remained as the fundamental technologies used for investigating and identifying the genetic disorders while illustrating the potential treatments and proper control of the disorder.
In conclusion, the future of cytogenetics remains bright.