Molecular Cytogenetic Techniques

Molecular cytogenetics is a discipline that combines molecular biology and cytogenetics and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956, when it was discovered that normal human cells contain 46 chromosomes. However, the first microscopic observations of chromosomes were reported by Arnold, Flemming, and Hansemann in the late 1800s, but their work was ignored for decades until the human chromosome number was determined to be 46. In 1879, Arnold examined sarcoma and carcinoma cells with very large nuclei. Today, the study of molecular cytogenetics is important for diagnosing and treating various malignancies, such as hematological malignancies, brain tumors, and other precancerous lesions. The field is generally concerned with the evolution of chromosomes, more specifically, the abnormalities in chromosome number, structure, function, and origin.

Fluorescent In Situ Hybridization (FISH)

FISH is a molecular cytogenetic technique that uses fluorescently labeled DNA or RNA probes to hybridize with target DNA sequences, thereby visualizing one or more specific regions of the genome under a microscope. FISH can be classified and improved according to different target sequences, probe designs, and hybridization methods to suit different research purposes. FISH can be performed on different types and stages of cells or tissues, such as metaphase or interphase chromosomes, nuclei, or tissue sections, or on the entire genome to detect copy number changes, a method called virtual karyotyping.

FISH has a wide range of applications in basic research and clinical diagnosis, mainly including the following aspects: gene mapping, which determines the location of genes or DNA fragments on chromosomes, thereby revealing their relationship with chromosome structure and function; chromosomal abnormalities, which detects numerical and structural abnormalities of chromosomes, such as trisomy syndromes, Edwards syndrome, Patau syndrome, chromosome deletions, duplications, inversions, translocations, etc.; gene expression, which detects the expression level and distribution pattern of specific genes or transcripts in cells or tissues, thereby revealing their relationship with cell function and development; genome evolution, which compares the genome structure and variation of different species or populations, thereby revealing their evolutionary history and phylogenetic relationship; cancer diagnosis, which detects the amplification, deletion or rearrangement of specific genes or chromosomal regions in cancer cells, thereby providing a basis for cancer diagnosis, typing, prognosis and treatment. FISH has the advantages of high sensitivity, high specificity, and high flexibility, but also has the disadvantages of complex operation, high cost, and difficult interpretation. To overcome these disadvantages, some improvement methods have been proposed, mainly involving probe design, hybridization methods, and signal detection.

Spectral Karyotyping (SKY)

KY is a molecular cytogenetic technique that uses a set of special probes to stain each chromosome with a whole-chromosome color, forming different colors of fluorescent signals. This set of probes consists of multiple small fragments of DNA probes, each of which is labeled with different proportions of three fluorescent dyes (red, green, and blue), resulting in different combinations of colors. When these probes hybridize with the target chromosomes, each chromosome can be observed with a unique color under a microscope. By performing spectral analysis and image reconstruction of the fluorescent signals, a full-color image of each chromosome can be obtained. SKY can be performed on metaphase chromosomes or on the entire genome to detect numerical and structural abnormalities of chromosomes, such as translocations, rearrangements, deletions, and duplications.

Fig.1 Spectral karyotyping (SKY) of the metaphase spread showing der(X)t(Xq;2p). (Raveendran S, 2015)

SKY has a wide range of applications in basic research and clinical diagnosis, mainly including the following aspects: chromosomal abnormalities, which detects numerical and structural abnormalities of chromosomes, such as trisomy syndromes, Edwards syndrome, Patau syndrome, chromosome deletions, duplications, inversions, translocations, etc.; cancer diagnosis, which detects the amplification, deletion or rearrangement of specific chromosomes or chromosomal regions in cancer cells, thereby providing a basis for cancer diagnosis, typing, prognosis and treatment; genome evolution, which compares the chromosome structure and variation of different species or populations, thereby revealing their evolutionary history and phylogenetic relationship; genome recombination, which detects artificially induced chromosome recombination, such as transgenic animals or cell lines. SKY has the advantages of high sensitivity, high specificity, and high convenience, but also has the disadvantages of high cost and difficult interpretation. To overcome these disadvantages, some improvement methods have been proposed, mainly involving probe design, signal detection, and image analysis.

Comparative Genomic Hybridization (CGH)

CGH is a molecular cytogenetic technique that uses DNA probes on a microarray chip to hybridize with target DNA sequences and detects copy number variations (CNVs) at the whole-genome level. CGH can compare two genomic DNA samples from different individuals or tissues and identify gains or losses of chromosomes or chromosomal regions without culturing cells.

CGH has various applications in basic research and clinical diagnosis, such as chromosomal abnormalities, cancer diagnosis, genome evolution, and genome recombination. CGH has the advantages of high resolution, wide coverage, and fast convenience, but also has the disadvantages of high cost and difficult interpretation. To overcome these disadvantages, some improvement methods have been proposed, involving chip design, signal detection, and data analysis. For example, using smaller and denser probes, more sensitive and stable fluorescent dyes, more accurate and fast scanners, and more powerful and intelligent computer software.

References

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