Journal: Journal of Histochemistry and Cytochemistry
Article Title: Bioinformatics Tools Allow Targeted Selection of Chromosome Enumeration Probes and Aneuploidy Detection
Figure Lengend Snippet: In situ hybridization results for selected DNA probes. Red shaded areas on the idiograms in (A–B) and (F–G) indicate the hybridization target sites. (A) In situ hybridization using fluorescent probes for the highly repeated satellite III DNA segment (flanked by the PCR primers WYR2 and WYR4) shows bright signals on the long arm of the Y chromosome. (B) Combining biotinylated in situ hybridization probes for the Y chromosome-specific DNA segment (large signal) with probes for X chromosome-specific DNA flanked by the PCR primers WXR1 and WXR2 (small signal) allows chromosome identification based on the size of the signal. (C) A digoxigenin-labeled DNA probe prepared from BAC RP11-348g24 resulted in specific signals on the X chromosome in the presence of a significant amount of cross-hybridization to other chromosomes in metaphase spreads. High level of cross-hybridization was also evident in interphase nuclei. (D) In contrast to the result shown in (C), a probe prepared from BAC RP11-294c12 bound almost exclusively to the centromeric heterochromatin of the human X chromosome. (E) Combining differentially labeled DNA probes in a single hybridization experiment, the Y chromosomal target appears in green, whereas the X chromosomal centromeric repeat DNA is shown in red. (F) In situ hybridization of an autosome-targeting DNA probe prepared from BAC clone RP11-96f8 showed multiple signals and a high level of cross-hybridization in the interphase cell nuclei. (G) The biotinylated probe prepared from BAC RP11-168p20 exhibited strong signals on both homologues of chromosome 10. (H) A combination of three differently labeled probes was hybridized simultaneously. Chromosome 10 signals are shown in red; X chromosomal and Y chromosomal signals are shown in green and blue, respectively. (I–K) The same probe as in (G) (BAC RP11-168p20) was hybridized to the cell line S48TK18A6. Multiple signals indicate chromosomal abnormalities. (I) shows DAPI picture, (J) shows green avidin-FITC signal, and (K) shows the superposition of both. (L–N) A chromosome X probe (BAC clone RP11-294c12) labeled with biotin/avidin-FITC, a Cy-5 labeled chromosome Y probe (RP11-242e13), and a chromosome 10-specific probe prepared from BAC RP11-168p20 (labeled with Spectral Orange-dUTP) were hybridized onto a deparaffinized 12-week placenta tissue section. (L) The chromosome 10 probe (red signals) and bound chromosome X probe (green signals) in this female placental tissue section. (M–N) In our triple probe FISH experiment, the chromosome 10 probe is represented by orange signals, the chromosome X probe by green signals, and the chromosome Y probe by red signals in the male cell nuclei. (Size marker bars indicate 10 µm.)
Article Snippet: DNA Probe Preparation The BAC DNAs were extracted from overnight cultures following an alkaline lysis protocol ( ) or using a ZR BAC DNA Miniprep Kit (Zymo Research; Irvine, CA).
Techniques: In Situ Hybridization, Hybridization, Polymerase Chain Reaction, Labeling, BAC Assay, Avidin-Biotin Assay, Fluorescence In Situ Hybridization, Marker