MOLECULAR CYTOGENETICS

Molecular Cytogenetics in the Era of Chromosomics and Cytogenomic

Approaches

I. INTRODUCTION
The current journal review mainly focuses on the molecular cytogenetics which
includes: the historical perspectives of its development from cytogenomics, technical
aspects, available probe sets, and the variants and applications of the basic
fluorescence in situ hybridization (FISH) approach. Wherein, The term "cytogenomics"
has become popular in place of the terms "cytogenetics" and/or "application of whole
genome oriented molecular genetic approaches." Wherein, to explain why changing
"molecular cytogenetics" to "molecular cytogenomics" is not justified in any way, even
though "molecular cytogenetics" is obviously a "cytogenomic approach," it is first
required to make a few comments on this subject.

i. Cytogenomics and Chromosomics

Although it was refered to as a broad term in 2019, cytogenomics merely reflects
technological advancements that fall within its purview, offering a shaky base for
displacing the established. the term cytogenetics is clearly defined. It was proposed that
cytogenomics be used to introduce the new integrative field of genomics and genetic
diagnostics that is comparable to chromosomics under this definition, and
chromosomics be used to concentrate on the three-dimensional morphology of
chromosomes, which is necessary for gene regulation and to summarize chromosome-
related studies in 2019.

II. CYTOGENETICS HISTORICAL ASPECT

Walter Flemming first identified chromosomes in 1879, Heinrich W. Waldeyer
later named Gregor Mendel's "linked up groups" as "chromosomes" in 1888, and Walter
Sutton and Theodor Boveri put forth the chromosome theory in 1902/03; however,
human cytogenetics evolved gradually, and Dr. Lore Zech first introduced banding
cytogenetics to detect changes in 1970. After C-banding and silver staining improved
cytogenetic techniques in the 1970s, GTG-banding became the gold standard for
chromosomal diagnosis. As a result, molecular cytogenetics was developed to 1)
capitalize on banding cytogenetics, but 2) overcome its low resolution, and 3) analyze
interphase cells. However, only radioactive ISH was feasible between 1969 and 1986,
and biotin was created as a nonradioactive hapten and the first

III. MOLECULAR CYTOGENETICS-FISH

FISH or Fluorescence in Situ Hybridization, initially available in single and dual

color, multicolor FISH has been around since 1998, making it the only molecular
cytogenetics technology still in use. First, all 24 human chromosomal paints were used
in a single experiment; as a result, a summary of other multicolor FISH probe sets is
provided elsewhere. With molecular combing becoming one of the most latest and
intriguing discoveries, FISH is used in research and diagnostics.
Numerous samples can be used for Molecular Cytogenetics. but chromosomal
preparation is necessary for banded cytogenetics. Both interphase nuclei and tissue
slices can be used for FISH. Target-DNA and probe-DNA are always required; the latter
must be marked with a hapten that can be seen under a fluorescence microscope, and
the following steps must be taken:
1. Target-DNA and probe-DNA denaturation.
2. Target- and probe-DNA were incubated in a hybridization solution at 37 °C for
several hours (with or without blocking of repetitive DNA sequences to avoid
background).
3. Removing extraneous probe-DNA by using the proper buffers.
4. If necessary, add antifade solution and a coverslip in order to detect hapten
bound to probe-DNA instead of using a fluorophore-labeled antibody.
Additional details are available elsewhere.

i. TYPES OF TARGET (DNA SAMPLES IN FISH)

FISH tests require intact, undamaged target DNA, which can come from pure
DNA of any species, native cells, extracted nuclei, tissue sections, or metaphase
chromosomes. Humans most frequently use readily available tissues like buccal
mucosa, bone marrow, skin fibroblasts, peripheral blood lymphocytes, and hair root
cells.

TYPES OF PROBE (DNA SUITED FOR FISH)

Commercial probes that are ready to use and labeled with fluorophores or
haptens are available for chromosomal research and diagnoses based on molecular
cytogenetics in humans. Five different types of FISH probe-DNA are listed below.
Labeling internal probes by PCR, Nick-translation, or the Universal Linkage System
(ULS) is necessary.
 LOCUS SPECIFIC PROBES
Locus-specific probes (LSPs) are typically cloned. Plasmids, artificial
chromosomes made from bacteria and yeast, and other genetic vectors with
species-specific DNA inserts spanning at least 12 kb are suitable. Contiguous
probes or single-copy probes can be used for mapping.
 REPETITIVE PROBES
Repeating DNA is found in FISH studies. Repeated probes that focus on
centromeres, telomeres, or satellite-DNA interspersion produce reliable
results that are simple to assess. At least one repetitive DNA (D424) can be
located using molecular methods in the number 4024.
 PARTIAL CHROMOSOME PAINTS
 Glass needle chromosomal microdissection is used to manufacture PCPS
(midi). A minimum of one or two euchromatic chromosomal subbands are
stained by PCPS, which are smaller than a chromosome arm.
 WHOLE CHROMOSOME PAINTS
Both midi and chromosomal flow sorting can be used to produce Whole
Chromosome Paints, or WCP. WCP probe sources also include interspecies
hybrids.
 WHOLE GENOME PROBES
Only when a technique is used—co-hybridizing two complete genomes
labeled in two different colors with typical human blood-derived metaphases
—can FISH make use of genomic DNA. This technology can be applied to
human genetic diagnostics using array-CGH or comparative cytogenomics
(CGH) (aCGH).
 MOLECULAR CYTOGENETICS PROBE SETS
Multicolor probe sets can be made with these FISH probes. In the following
sections, a select few probe sets and their applications in cytogenomic
techniques are covered.
IV. MOLECULAR CYTOGENETICS IN ROUTINE DIAGNOSTICS
Molecular cytogenetics is essential in many areas of genetics, including
prenatal, postnatal, and tumor diagnostics on cytogenetically worked-up cells, and
FISH is also used to identify solid tumors in pathology using FFPE material, despite
difficulties with reimbursement for routine FISH-diagnostics. All two- to multi-color
FISH probes are used in routine molecular cytogenetic diagnostics, so the number
and kind of probes used in metaphase-FISH are unlimited, whereas interphase-FISH
uses fewer than six LSPs and/or centromeric probes. In the last ten years, probe
sets have been developed to identify fusion partners and/or detect inversions in
interphase nuclei in addition to copy number loss or gain, translocations, gene
fusions, or gene splitting.
V. MOLECULAR CYTOGENETIC APPLICATIONS FOR CHROMOSOMIC
RESEARCH IN THE CONCERT OF CYTOGENOMICS APPROACHES
This choice of research and diagnostic areas is arbitrary because some of the
approaches listed below can characterize DNA segments with several to hundreds
of base pairs, while others focus on chromosomal subregions, bands, or entire
chromosomes; still others provide information at the genome level. Molecular
cytogenetics applications in plant research or microorganisms are not coated,
despite the fact that molecular cytogenetics is one of two cytogenomic methods that
can analyze entire genomes from the base pair to the chromosome level. This is due
to the large number of FISH applications and the author's emphasis on human
genetics.
 MOLECULAR COMBING
High-molecular-weight DNA was physically combed on a glass surface in
1994; molecular combed is now available on the market, allowing research on
most stretched DNA-fibers: FISH probes can be hybridized, and basic studies
on DNA-replication, replication kinetics, but also copy number variations of
satellite sequences down to SNPs can be visualized. By using molecular
combining, it is simple to distinguish between the D4Z4 sequences on 4q35
and 10q26 for FSHD diagnoses. High-resolution FISH is expected to advance
further.
 CHROMOSOME ORIENTATION- FISH (CO-FISH)
5-Bromodeoxyuridine (BrdU) is integrated into DNA as a result of CO-FISH
labeling the chromosome's DNA strand, where it is then broken down by UV
light and EXOIII (the latter detects UV-induced gaps and starts degradation of
the DNA strand there). Long or repetitive DNA sequences are examined
using CO-FISH. The FISH-benefits methods have not been fully examined.
 TELOMERES ACCESSED BY Q-FISH
Sequencing the low-copy repeating components of telomeres is difficult.
Some techniques for determining telomere length include qPCR, terminal
restriction fragment analysis, telomere dysfunctional caused foci analysis,
single telomere length analysis, telomere shortest length assay, and
quantitative FISH (Q-FISH).
 PARENTAL ORIGIN DETERMINATION
The undetectable CNVs in humans include deletions, gains, insertions, and
inversions. Human CNVs were first discovered in 2001 as a result of a large-
scale investigation using aCGH, which is now known as CMA (chromosomal
micro-array). Inferring that microsatellites track father-mother-child
chromosomes while POD FISH shows chromosome inheritance and
microsatellites cannot divide chromosomes is possible because each CNV
has a unique pattern that can be used to distinguish between homologous
chromosomes.
 INTER AND INTRA CHROMOSOMAL INTERACTIONS
Two methods are used to investigate chromosome interactions: high-
throughput chromosome conformation capture is used in multiple-cell
genomics, and high-throughput sequencing enhances genomic architecture
by discovering chromatin loci pairs. Both single-cell Hi-C and imaging 3-D
genome structure, which allow for many chromatin loci per cell, as well as
these data do not allow for the identification of 3D alleles and loci;
nonetheless, both techniques found TADs and intra- and interchromosomal
connections.
 MULTI-COLOR FISH IN RESEARCH
Multiplex-FISH (M-FISH) and spectral karyotyping (SKY) started routinely
painting all 24 human chromosomes with complete probes, and FISH tests
spread as a result. Multicolor-FISH (mFISH) techniques and probe sets are
reimbursed by health systems for routine diagnostics. Murine multicolor
banding (mcb) was used to characterize chromosome evolution and tumor
cell lines, while most mFISH-probe sets were used to diagnose inherited or
acquired chromosomal abnormalities.
 RESEARCH ON SMALL SUPERNUMERARY MARKER
CHROMOSOMES
Since there are 3.3 million asymptomatic human sSMC carriers worldwide,
similar to B-chromosomes in other species, and since normal sSMC carriers
can have partial tri- or tetrasomies near centromeres, molecular cytogenetics
is the most direct method for identifying the genetic makeup of sSMCs.
SSMCs are frequently missed by molecular karyotyping or sequencing due to
their low mosaic and/or heterochromatic states, and centromere-specific
multicolor FISH determines the sSMC's origin (cenM-FISH).
 CHROMOANAGENESIS RESEARCH
Chromosomal rearrangements have been studied for decades by
cytogenetics and molecular cytogenetics, with the latter revealing single cells
with altered chromosomes and/or chromosome pulverization. In 2011,
chromothripsis was "rediscovered" as a result of NGS research; since then,
chromothripsis, chromoanasynthesis, and chromoplexy have all been
combined under the term "chromothripsis," and new chromothripsis studies
combine NGS with molecular cytogenetics.
 CHROMOSOMALS HETEROMORPHISMS AND REPITITIVE DNA
ELEMENTS
Chromosome heteromorphisms (CHMs) are only accessible through
cytogenetics and molecular cytogenetics, and recurrent CHMs, like satellite
DNAs, are poorly understood. DXZ1 and DYZ3 are commercially available
centromere-specific probes for chromosomes X and Y because no genome
browser contains satellite DNAs from the 1980s in human pericentric and/or
heterochromatic regions.
REFLECTION:

The study of chromosomal and subchromosomal genome variation is known as

"cytogenomics." In that it concentrates on the three-dimensional morphology of
chromosomes for gene regulation, cytogenomics is comparable to
chromosomics. All chromosome-related studies should be compiled under one
umbrella term in order to introduce innovative ideas and concepts in biology and
medicine. Banding cytogenetics can be used to study interphase cells, but
molecular cytogenetics was developed to improve on its low resolution. The only
molecular cytogenetics technique still in use, FISH, was the first to use single-
and dual-color fluorescent probes.
Numerous samples can be utilized for molecular cytogenetics. The disciplines of
subjectivity and diagnosis were selected. Given the numerous FISH applications
and the author's emphasis on human genetics, this is the case. Due to the fact
that the normal FISH resolution is kilo- to megabase pair (except for molecular
combing), exact mapping of chromosomal breakpoints requires the use of
numerous methods.
FISH, as well as cytogenetics and next-generation sequencing, cannot detect
disease-causing gene mutations at the base-pair level (NGS). Experienced
specialists are required for qualified evaluation and interpretation of FISH results,
replenishable costs may be high, and the number of probes used concurrently is
limited by fluorochromes and software; however, recent developments offer
solutions. As molecular cytogenetics does not yet permit intricate and costly
approaches, it is anticipated that advancements will be made. Overall, current
diagnosis and research continue to rely heavily on molecular cytogenetics.