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Chromosome Research

, Volume 26, Issue 4, pp 307–315 | Cite as

A combined banding method that allows the reliable identification of chromosomes as well as differentiation of AT- and GC-rich heterochromatin

  • Natalya A. LemskayaEmail author
  • Anastasia I. Kulemzina
  • Violetta R. Beklemisheva
  • Larisa S. Biltueva
  • Anastasia A. Proskuryakova
  • John M. Hallenbeck
  • Polina L. Perelman
  • Alexander S. Graphodatsky
Original Article
  • 223 Downloads

Abstract

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.

Keywords

Сonstitutive heterochromatin Differential staining Heterochromatin composition Karyotype Chromosome C-banding G-banding AT-rich GC-rich 

Abbreviations

αMEM

Alpha minimum essential medium

ATrH

AT-rich heterochromatin

CBG-banding

C-bands revealed by barium hydroxide treatment followed by Giemsa staining

FISH

Fluorescence in situ hybridization

CDAG

Chromomycin A3-DAPI-after G-banding

Cen

centromere

CMA3

chromomycin A3

DABCO

1,4-diazabicyclo[2,2,2]-octane

DAPI

4′-6-diamidino-2-phenylindole

DIPI

4-6-bis-(2-imidozolynyl-4H,5H)-2-phenylindole

DMSO

Dimethyl sulfoxide

DPBS

Dulbecco’s phosphate-buffered saline

EDTA

Ethylenediaminetetraacetic acid

FBS

Fetal bovine serum

GCrH

GC-rich heterochromatin

GTG-banding (G-banding)

G-bands by trypsin followed by Giemsa staining

H

heterochromatin

PBS

Phosphate-buffered saline

RT

room temperature

o/n

overnight

Notes

Acknowledgments

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.

Author’s contribution

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.

Funding information

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.

Supplementary material

10577_2018_9589_MOESM1_ESM.docx (13 kb)
ESM 1 (DOCX 13 kb)

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Natalya A. Lemskaya
    • 1
    Email author
  • Anastasia I. Kulemzina
    • 1
  • Violetta R. Beklemisheva
    • 1
  • Larisa S. Biltueva
    • 1
  • Anastasia A. Proskuryakova
    • 1
    • 2
  • John M. Hallenbeck
    • 3
  • Polina L. Perelman
    • 1
    • 2
  • Alexander S. Graphodatsky
    • 1
    • 2
  1. 1.Institute of Molecular and Cellular Biology SB RASNovosibirskRussia
  2. 2.Novosibirsk State UniversityNovosibirskRussia
  3. 3.Stroke BranchNational Institute of Neurological Disorders and Stroke, National Institutes of Health (NINDS/NIH)BethesdaUSA

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