Protocol detail

Centromeric Repeat Sequence Analysis

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FISH and fiber-FISH were carried out according to published protocols41 (link)61 (link)62 (link). The DNAs labeled with digoxigenin-dUTP (Roche Diagnostics, USA) and Biotin-dUTP (Roche Diagnostics, USA) were detected using rhodamine-conjugated anti-digoxigenin (Roche Diagnostics, USA) and fluorescein-conjugated avidin (Life Technologies, USA), respectively. DNAs were labeled with digoxigenin-dUTP and Biotin-dUTP for FISH analysis. Slides were examined under Olympus BX63 fluorescence microscope (Olympus, Japan). Chromosome and signal images were captured and merged using CellSens Dimension software (Olympus, Japan). Fiber-FISH was conducted to reveal the organization of the centromeric repeat sequences in SES208 genome. The fiber-FISH signals were measured and converted into kb using a 3.21-kb/μm conversion rate41 (link).

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Zhang W., Zuo S., Li Z., Meng Z., Han J., Song J., Pan Y.B, & Wang K. (2017). Isolation and characterization of centromeric repetitive DNA sequences in Saccharum spontaneum. Scientific Reports, 7, 41659.

Publication 2017

digoxigenin-dUTP Biotin-dUTP rhodamine-conjugated anti-digoxigenin fluorescein-conjugated avidin BX63 fluorescence microscope CellSens Dimension software

Biotin Centromeric Chromosome Diagnostics Digoxigenin Dnas Dutp Fiber Fish Fluorescein avidin Fluorescence microscope Genome Repeat sequences Rhodamine

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Protocol cited in 1 other protocol

Variable analysis

independent variables

Labeling of DNA with digoxigenin-dUTP and Biotin-dUTP

dependent variables

Detection of labeled DNA using rhodamine-conjugated anti-digoxigenin and fluorescein-conjugated avidin
Measurement of fiber-FISH signals and conversion to kb

control variables

Published protocols referenced for FISH and fiber-FISH procedures (41, 61, 62)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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