Bioinformatic Tools Identify Chromosome-Specific DNA Probes and Facilitate Risk Assessment by Detecting Aneusomies in Extra-embryonic Tissues

Abstract

Despite their non-diseased nature, healthy human tissues may show a surprisingly large fraction of aneusomic or aneuploid cells. We have shown previously that hybridization of three to six non-isotopically labeled, chromosome-specific DNA probes reveals different proportions of aneuploid cells in individual compartments of the human placenta and the uterine wall. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes. Chromosome losses in placental or uterine tissues, on the other hand, were detected infrequently. A more thorough numerical analysis of all possible aneusomies occurring in these tissues and the investigation of their spatial as well as temporal distribution would further our understanding of the underlying biology, but it is hampered by the high cost of and limited access to DNA probes. Furthermore, multiplexing assays are difficult to set up with commercially available probes due to limited choices of probe labels. Many laboratories therefore attempt to develop their own DNA probe sets, often duplicating cloning and screening efforts underway elsewhere. In this review, we discuss the conventional approaches to the preparation of chromosome-specific DNA probes followed by a description of our approach using state-of-the-art bioinformatics and molecular biology tools for probe identification and manufacture. Novel probes that target gonosomes as well as two autosomes are presented as examples of rapid and inexpensive preparation of highly specific DNA probes for applications in placenta research and perinatal diagnostics.

Bioinformatics Tools Allow Targeted Selection of Chromosome Enumeration Probes and Aneuploidy Detection

“…Chromosome X and Y BAC clone selection and validation was previously described by Zeng et al (2011, 2012). A chromosome-specific 3.5-kb satellite III DNA repeat had been identified (Nakahori et al 1986) in the q12 band of chromosome Y, for which we designed and tested various PCR primer sets (Zeng et al 2011).…”

Full Karyotype Interphase Cell Analysis

“…During preparation of this manuscript, Ioannou et al demonstrated this, using BAC clones from Roswell Park Cancer Institute RP11 library. We decided to combine preexisting DNA repeat probes with optimized BAC contigs, which were identified using bioinformatics 22,42 to obtain optimal specificity and brightness (Table 2). Now, combining the versatility of BACs and preexisting repeat probes with a wide repertoire of different fluorochromes, together with the use of the SKY system, resulted in a cost-effective, flexible, and reliable methodology to detect four sets of six probes in interphase nuclei (see Figs.…”

“…During preparation of this manuscript, Ioannou et al demonstrated this, using BAC clones from Roswell Park Cancer Institute RP11 library. We decided to combine preexisting DNA repeat probes with optimized BAC contigs, which were identified using bioinformatics 22,42 to obtain optimal specificity and brightness (Table 2).…”

High Performance DNA Probes for Perinatal Detection of Numerical Chromosome Aberrations

“…The probe, when hybridized to metaphase spreads prepared from the fibroblast cell line WI-38 and stained with avidin-FITC following our published protocol [ 24 ], showed two very bright, specific signals per cell (arrows in Figure 2B ) which were easy to count in the microscope by eye. Other probes, which have been prepared using a similar bioinformatics approach for chromosomes 10, X and Y can easily be combined with this chromosome 16-specific probe [ 50 – 52 ].…”