Comparative Analysis of Three Methods to Produce Diagnostic FISH Probes: Nick Translation, PCR Amplification and Chromosome Microdissection

Document Type : Original Article

Authors

National Research Centre

Abstract

Introduction: Fluorescence in situ hybridization (FISH) probes are vital diagnostic tools used to identify genetic disorders and malignancies. These probes enable precise localization of specific DNA sequences within chromosomes, making them crucial for both research and clinical diagnostics. Aim: The aim of this study is to evaluate and compare the effectiveness of three distinct methods for generating FISH probes by assessing the quality, advantages, and limitations of each. We aimed to provide insights into their suitability for producing locus-specific identifier and whole chromosome paint probes for diagnostic applications. Methods: In all used methods we followed the guidelines established by the American College of Medical Genetics (ACMG) for FISH probe production. We employed three methods to generate diagnostic probes: 1- Nick Translation to incorporate the labeled dyes into DNA segments from BAC clones or chromosome-specific DNA as templates. 2- PCR Amplification and Labeling using genomic DNA as a template and specific primers 3- Chromosome Microdissection using laser dissection microscope (LDM) to dissect and isolate specific chromosomes for further amplification. Results: Using BAC clones and nick translation, we developed locus-specific probes for chromosomes 6p, 6q, 1p36, and PAR1. Furthermore, by using chromosome-specific DNA templates and nick translation, we successfully produced WCPs for chromosomes 1, 4, 6, 14, 22, and X. PCR amplification and Labeling Using specific primers, we successfully produced alpha-satellite probes for several chromosome centromeres which were published before. Degenerate oligonucleotide-primed PCR (DOP-PCR) was employed to amplify a whole chromosome paint (WCP) for chromosome 13. Chromosome microdissection enabled the production of chromosome 6 centromere; however, amplifying small DNA quantities remains a challenge. Conclusion: Nick translation is the most effective and generative method for producing locus-specific probes and WCPs due to its reproducibility, accuracy, and stability. PCR-based methods are efficient for short unique repetitive sequences of chromosomes centromere. DOP-PCR is effective for amplification of entire chromosomes. While chromosome microdissection is promising, optimizing DNA amplification from very small DNA quantity is critical. The use of BAC clones ensures a sustainable resource for developing multiple locus-specific and whole chromosome paint probes, significantly addressing our research needs.

Keywords

Files

Share

How to cite

Statistics