A combined banding method that allows the reliable identification of chromosomes as well as differentiation of AT- and GC-rich heterochromatin
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Сonstitutive heterochromatin areas are revealed by differential staining as C-positive chromosomal regions. These C-positive bands may greatly vary by location, size, and nucleotide composition. CBG-banding is the most commonly used method to detect structural heterochromatin in animals. The difficulty in identification of individual chromosomes represents an unresolved problem of this method as the body of the chromosome is stained uniformly and does not have banding pattern beyond C-bands. Here, we present the method that we called CDAG for sequential heterochromatin staining after differential GTG-banding. The method uses G-banding followed by heat denaturation in the presence of formamide with consecutive fluorochrome staining. The new technique is valid for the concurrent revealing of heterochromatin position due to differential banding of chromosomes and heterochromatin composition (AT-/GC-rich) in animal karyotyping.
KeywordsСonstitutive heterochromatin Differential staining Heterochromatin composition Karyotype Chromosome C-banding G-banding AT-rich GC-rich
Alpha minimum essential medium
C-bands revealed by barium hydroxide treatment followed by Giemsa staining
Fluorescence in situ hybridization
Chromomycin A3-DAPI-after G-banding
Dulbecco’s phosphate-buffered saline
Fetal bovine serum
- GTG-banding (G-banding)
G-bands by trypsin followed by Giemsa staining
We would like to acknowledge Dr. David McMullen for providing the tissue for establishing the cell line used in this study. We acknowledge anonymous reviewers and the editor whose comments helped to improve the manuscript. We are thankful to Dr. Tatyana Kolesnikova for helpful discussion.
NAL conceived, designed, performed research, and analyzed data. NAL, AIK, VRB, LSB, AAP, PLP conducted experiments. ASG, VRB analyzed data. JMH provided the critical sample. NAL wrote the manuscript. PLP, VRB, LSB edited manuscript. All authors read and approved the manuscript.
The work was supported by the Russian Science Foundation (RSF, 16-14-10009). The work on human karyotypes was supported by RFBR according to the research project No. 18-04-00826.
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