Atomic Force Microscopic Examination of Chromosomes Treated with Trypsin or Ethidium Bromide

Abstract

Recently, researchers have sought to better understand the relationship between chromosomal structure and function. Chromosomes, primarily composed of DNA and histone proteins, must replicate prior to cell division. During replication, errors can be introduced into the DNA via physical or chemical stressors, including heat, ultraviolet radiation, and other forms of radiation, heavy metal ions, drugs and organic reagents.1,2) However, it is also probable that chromosomal alterations might be introduced outside of replication, such as during the trypsinization or ethidium bromide (EB) staining processes 3) commonly used in visualization of chromosomes by optical microscopy. Changes of chromosome ultrastructure cannot be observed clearly under conventional optical microscopes due to technical difficulties such as diffraction limitations. However, the advent of atomic force microscopy (AFM) 4) has allowed researchers to investigate small alterations in chromosome structure and morphology.Previous studies have examined changes in chromosome structure caused by physicochemical treatments.5,6) Acetic acid, which is frequently used to remove cellular contaminants, was applied to barley chromosomes at concentrations of 15, 30 and 45%. AFM analysis revealed that the optimal concentration was 30%; at lower levels sufficiently clean chromosomes samples could not obtain and the chromosome surface structures could not be observed, and at higher levels the chromosome structures were significantly damaged, although the cellular contaminants were effectively removed. 7)Similar studies identified chromosomal damages induced by treatments with NaCl 8) and heavy ion radiation. 9)EB, a heterocyclic organic compound, can insert between base pairs, 10) resulting in increasing DNA rigidity and lengthening of DNA.11) Low doses of EB can induce DNA denaturation and can lead to cancer formation. 12) Thus, it is possible that EB staining of chromosome preparations may induce structural abnormalities 13) not present in the actual organism. Up to now, though there are some works about structural changes of chromosomes caused by physicochemical treatments, 3,5,6,[14][15][16] however, no previous work has used AFM to examine the effects of time-course of trypsin or different dose EB on chromosomal structures.In this article, we used AFM in the tapping mode to examine chromosomal damage induced by EB treatment and trypsin digestion in air. Our results showed that EB could profoundly damage human chromosomes, leading to breakages and separations in a dose-dependent fashion. We also found that trypsinization could obviously change the morphological features of chromosomes with increased digestion time; the optimal digestion time for AFM analysis appeared to be 10-20 s under our experimental conditions, but not to be over 40 s. In addition, our results indicate that AFM is a powerful tool for studying chromosomal damage, perhaps indicating that it will be applicable to further diagnostic applications. Together, our results indicate that common chr...

Cytogenetic stability of chicken T‐cell line transformed with Marek's disease virus: atomic force microscope, a new tool for investigation

“…The use of AFM is advantageous because no enzymatic treatment such as with trypsin is needed to evidence the heterochromatic and euchromatic regions of chromosomes. The treatments with trypsin damage the structure of chromosomes thus probably affecting the observed results (Wu et al ., 2006). The topographical data obtained with AFM, without trypsin treatment, are basically needed to identify the different chromosomal regions and allow the recognition of duplications and deletions presented in this study.…”