title: Replication Banding Patterns in Human Chromosomes Detected Using 5-ethynyl-2'-deoxyuridine Incorporation.
abstract: A novel technique using the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into replicating DNA is described for the analysis of replicating banding patterns of human metaphase chromosomes. Human lymphocytes were synchronized with excess thymidine and treated with EdU during the late S phase of the cell cycle. The incorporated EdU was then detected in metaphase chromosomes using Alexa Fluor® 488 azides, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acetylene group of EdU. Chromosomes with incorporated EdU showed a banding pattern similar to G-banding of normal human chromosomes. Imaging by atomic force microscopy (AFM) in liquid conditions showed that the structure of the chromosomes was well preserved even after EdU treatment. Comparison between fluorescence microscopy and AFM images of the same chromosome 1 indicated the presence of ridges and grooves in the chromatid arm, features that have been previously reported in relation to G-banding. These results suggest an intimate relationship between EdU-induced replication bands and G- or R-bands in human chromosomes. This technique is thus useful for analyzing the structure of chromosomes in relation to their banding patterns following DNA replication in the S phase.
authors.affiliation: Anatomy and Physiological Science, Graduate School of Health Care Science, Tokyo Medical and Dental University, Tokyo 113-8519, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Hoshi
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Ushiki
references.citation: J Org Chem. 2002 May 3;67(9):3057-64
references.pmid: 11975567
references.citation: Brain Res. 2010 Mar 10;1319:21-32
references.pmid: 20064490
references.citation: Proc Natl Acad Sci U S A. 1973 Dec;70(12):3395-9
references.pmid: 4128545
references.citation: Chromosoma. 1989 Sep;98(3):174-80
references.pmid: 2582898
references.citation: Methods Mol Biol. 2011;736:109-15
references.pmid: 21660724
references.citation: Angew Chem Int Ed Engl. 2002 Jul 15;41(14):2596-9
references.pmid: 12203546
references.citation: Cytobios. 1988;56(225):107-25
references.pmid: 3251682
references.citation: J Neurosci Methods. 2009 Feb 15;177(1):122-30
references.pmid: 18996411
references.citation: Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2415-20
references.pmid: 18272492
references.citation: Arch Histol Cytol. 2001 Dec;64(5):475-82
references.pmid: 11838707
references.citation: Chromosome Res. 2008;16(3):383-96
references.pmid: 18461479
references.citation: Cytogenet Cell Genet. 1996;75(2-3):172-9
references.pmid: 9040787
references.citation: Cytogenet Genome Res. 2004;107(1-2):28-31
references.pmid: 15305053
references.citation: Nucleic Acids Res. 1990 May 11;18(9):2831
references.pmid: 2339076
references.citation: Arch Histol Cytol. 2002 Dec;65(5):377-90
references.pmid: 12680454
references.citation: Biotechniques. 2008 Jun;44(7):927-9
references.pmid: 18533904