Direct detection of alpha satellite DNA with single-base resolution by using abasic Peptide Nucleic Acids and Fluorescent in situ Hybridization.

The detection of repetitive sequences with single-base resolution is becoming increasingly important aiming to understand the biological implications of genomic variation in these sequences. However, there is a lack of techniques to experimentally validate sequencing data from repetitive sequences obtained by Next-Generation Sequencing methods, especially in the case of Single-Nucleotide Variations (SNVs). That is one of the reasons why repetitive sequences have been poorly studied and excluded from most genomic studies. Therefore, in addition to sequencing data, there is an urgent need for efficient validation methods of genomic variation in these sequences. Herein we report the development of chemFISH, an alternative method for the detection of SNVs in repetitive sequences. ChemFISH is an innovative method based on dynamic chemistry labelling and abasic Peptide Nucleic Acid (PNA) probes to detect in situ the α-satellite DNA, organized in tandem repeats, with single-base resolution in a direct and rapid reaction. With this approach, we detected by microscopy the α-satellite DNA in a variety of human cell lines, we quantified the detection showing a low coefficient of variation among samples (13.16%-25.33%) and we detected single-base specificity with high sensitivity (82.41%-88.82%). These results indicate that chemFISH can serve as a rapid method to validate previously detected SNVs in sequencing data, as well as to find novel SNVs in repetitive sequences. Furthermore, the versatile chemistry behind chemFISH can lead to develop novel molecular assays for the in situ detection of nucleic acids.

Development of a nanotechnology-based approach for capturing and detecting nucleic acids by using flow cytometry.

Nucleic acid-based molecular diagnosis has gained special importance for the detection and early diagnosis of genetic diseases as well as for the control of infectious disease outbreaks. The development of systems that allow for the detection and analysis of nucleic acids in a low-cost and easy-to-use way is of great importance. In this context, we present a combination of a nanotechnology-based approach with the already validated dynamic chemical labeling (DCL) technology, capable of reading nucleic acids with single-base resolution. This system allows for the detection of biotinylated molecular products followed by simple detection using a standard flow cytometer, a widely used platform in clinical and molecular laboratories, and therefore, is easy to implement. This proof-of-concept assay has been developed to detect mutations in KRAS codon 12, as these mutations are highly important in cancer development and cancer treatments.