**4.9 High resolution banding**

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

as useful for visualization and study of satellite associations.

The NOR regions could be selectively stained by techniques involving either giemsa or silver staining. The giemsa technique developed by Matsui and Sasaki [29] allows the staining NOR after extraction of nucleic acids and histones. The technique N banding was improved by Funaki et al. [30]. The silver staining technique fall into two categories; a) Ag-As method: the method is based on the staining with combined silver nitrate and ammoniacal silver solutions; b) Ag method: in this method, staining with ammoniacal silver is omitted. NOR banding stains only those regions that were active as nucleolus organizers in the preceding interphase as well

In routine cytogenetic diagnosis, a single banding technique is usually sufficient for the detection of chromosomal abnormalities e.g. G banding or R banding, but sometimes, more complicated chromosomal rearrangements often require sequential staining of the same metaphase by several banding techniques and the process is known as sequential banding. The quality of chromosomes in sequential banding deteriorates with each staining therefore; it restricts the sequential banding up to 3 or 4 different staining techniques (**Figure 5**). For example, single metaphase ➔ First procedure, Q banding ➔ Second procedure, G banding ➔ Third procedure, C banding ➔ deteriorates the chromosome quality ➔ therefore, restricts up to 3 or 4

This is the technique that produces simultaneously two types of banding on the same metaphase or on one slide but different metaphases (**Figure 5**). For example, same metaphase ➔ first procedure, G banding ➔ second procedure, C banding OR single slide with different metaphases ➔ first procedure, C banding ➔ second procedure, T banding. Simultaneous banding restricts up to two staining procedures in different or single metaphase and results in precise estimation of chromosomal

**4.6 N (Nucleolar organizing regions) banding**

**4.7 Sequential banding**

staining procedures [31].

aberrations [32].

**4.8 Simultaneous banding**

**52**

**Figure 5.**

*Banding techniques for sequential and simultaneous staining.*

The general trend is to pick highly thick and contracted metaphase chromosomes are chosen for banding procedures which gives enough banding patterns for prediction of any kind of aberrations or arrangements within complement, but sometimes it fails also. Therefore, elongated chromosomes (earlier stages of mitotic divisions before reaching metaphase stage) are standardized for banding patterns and prediction of aberrations or arrangements called high resolution banding. Elongated chromosome standardization and preparation could be obtained through synchronization of cell cycle or use of various chromosome anti-contraction reagents through a procedure known as cell cycle synchronization technique. Cell synchronization is a process by which mitotic cells at different stages of cell cycle in culture are brought to the same phase through physical fractionation or chemical blockage or inhibition of DNA synthesis during S-phase. Cell synchrony may be defined as the progression of cells through cell cycle. The possible procedure for high resolution banding pattern are described. Mitotic stages (cultured cells) ➔ cell synchronization by adding chemical reagents ➔ Amethopterin or methotrexate or thymidine or fluorouracil ➔ cultured cells ➔ Blocks DNA synthesis in cultured cells ➔accumulation of cells in S-phase of cell cycle ➔ block released ➔ cell synchrony (large quantities of cells continue their cycle from the same level) ➔ prophase to mid-metaphase range ➔ high number of bands (gives more information as compared to the compact metaphase banding) [33, 34].

The technique allows precise localization of break points in chromosomal rearrangements and detection of minute chromosomal alterations that are undetectable by the mid-metaphase banding techniques [35].

#### **5. Chromosome differentiation**

Conventional chromosome banding techniques help us in understanding the patterns associated with chromosomal evolution of a species, speciation processes (formation of a new species) and generation of genetic variability among the species. Similarly, molecular banding techniques such as FISH and GISH provide little more and specific information by causing more differentiation in banding pattern of a chromosome of a particular species. Now, computational analyses (software packages) of the chromosome bands provide maximum information on banding pattern by increasing number of bands which helps to predict the specific and precise result on chromosomal aberrations and arrangements (**Figure 6**). For example, conventional chromosome bands ➔ enough bands for analysis ➔ molecular banding techniques ➔ more bands, more differentiation, more information ➔ computational techniques (software packages) ➔ still more bands, still more differentiation, still more information [36].

#### **6. Chromosome linearization**

Chromosome linearization is an important tool under computational analyses of the chromosome bands which suggest that linear chromosomes will provide maximum number of bands as well as information regarding aberrations or arrangements as compared to the twisted, rounded, curved and overlapped chromosomes. The information obtained from the straight chromosomes will be

## *Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

larger or maximum in quantity and quality. The tool of image linearization enables a better visualization technique which ultimately extends and refines the information that can be extracted from the chromosomes (**Figure 6**). For example, conventional chromosome bands ➔ enough bands for analysis ➔ molecular banding techniques ➔ more bands, more differentiation, more information ➔ computational techniques (software packages) ➔ better organization of cytogenetic data ➔ tool of image linearization ➔ still more bands, still more differentiation, still more information [37].

**Figure 6.**

*Characteristics of chromosome linearization and differentiation banding technique.*

**55**

*Chromosome Banding and Mechanism of Chromosome Aberrations*

application of different staining and banding procedures [39].

**8. Detection and analysis of preliminary chromosomal aberrations**

The primary chromosome aberrations could be detected and analysed in three forms such as achromatic lesions, chromosome type and chromatid type structural aberrations. The detection and characterization of aberrations or breaks which falls in the light banded region or euchromatic heterochromatin region which generally takes light stain could be identified as achromatic lesion. Chromosome type structural aberrations could be detected and identified in the form of asymmetrical interchanges (two translocations in the same arm), symmetrical interchanges (reciprocal translocations), inter arm intra changes (centric ring, pericentric inversions), intra arm intra changes (interstitial deletions, paracentric inversions), and breaks [(i) fragment compound (usually contains two terminal regions) and suspects an incomplete dicentric or centric ring; compound fragment with a terminal and unrelated non-terminal region suspects an incomplete complex interchanges, (ii) fragment simple (genetic material from one arm only) but with altered banding sequencing, suggest that sequence alteration is most frequently cause the inversion of a chromosomal proximal segment which is most probably indicates an incomplete paracentric inversion, (iii) fragment simple but the normal band sequence; (a) short arm with normal band sequences, if fragment present and not related to short arm might be incomplete reciprocal translocation or pericentric inversion; if fragment present and related to short arm may be true terminal deletion; (b) short arm with abnormal band sequences, first, observe for a distal inversion or incomplete paracentric inversion, second, observe for distal genetic

The chromosome banding primarily could be used for the detection and recognition of nature and type of the aberrations, identification of the chromosome involved and most importantly, the location of the presumptive positions of the lesions (usually termed as break points) involved in the structural changes in

The basic requirement for the detection of chromosomal structural changes using the banding methods is disruption in the normal sequential band pattern of a chromosome arm region. The disruption in the chromosomal arm may take several forms and the most common forms are presence of an additional band, absence of an expected band, unexpected change in banding pattern and reversion of a part of banding pattern. The basic requirements for the chromosomal structural changes are possible by the existence of consistent and fixed pattern of chromosomal bands on chromosomal arm region as well as existence of good sequential differentiation between the chromosomal arm bands. Chromosome condensation is a process that occurs as the cell progresses towards metaphase and continues till the cells are held at metaphase by the action of spindle inhibiting chemicals. Consequently, number of bands, banding pattern, clarity and differentiation among the bands would be a function of state of chromosome contraction and possible provides the existence of consistent and fixed pattern of chromosomal bands at metaphase on chromosomal arm region. The good sequential differentiation between the chromosomal arm bands may cause differences in size, staining intensity, longitudinal spacing, numerical concentration of dark and light bands which forms the basis for the identification of individual chromosomes, but sometimes, the quality of differentiation varies between treatments and also between the cells on the same slide by

*DOI: http://dx.doi.org/10.5772/intechopen.96242*

**7. Chromosome banding application**

chromosome complements [38].
