FISH with Padlock Probes Can Efficiently Reveal the Genomic Position of Low or Single-Copy DNA Sequences

Abstract

A padlock probe is a circularized oligonucleotide containing complementary sequences of target regions. Padlock probes combined with rolling-circle amplification enable fluorescence in situ hybridization (FISH) to reproducibly detect the genomic position of low or single-copy DNA sequences. The FISH with padlock probes revealed that a plant gene cluster of tandemly arrayed three paralogues responsible for photosynthesis rapidly moved from the interior subnuclear region to the peripheral subnuclear region in response to light. Using a pair of peptide nucleic acids as a double-strand opener for the target region, the efficiency of FISH with padlock probes at the detection of a single-copy genomic region was much improved. FISH with padlock probes will give us reliable data of dynamics, arrangement and position of a specific single locus in both the nucleus and the condensed chromosomes.

Various Strategies for Improved Signal-to-Noise Ratio in CRISPR-Based Live Cell Imaging

“…To study the regulation of chromatin dynamics in living cells, imaging strategies to detect a specific genomic site or gene locus has been developed Matsunaga 2016, Hirakawa andMatsunaga 2016). Classical imaging methods, such as fluorescent in situ hybridization (FISH), have excellent signal-to-noise ratios but require pre-treatment of cells, such as the cell fixation and the DNA denaturation, which prevents the capture of chromatin dynamics (Matsunaga 2016, Matsunaga and Matsunaga 2017, Kikuchi and Iwamoto 2020, Shafiee et al 2020, Suto et al 2020, Widarmi et al 2020, Amiad-Pavlov et al 2021, Hondo and Azumi 2021, Suto et al 2021, Wang and Sheng 2022. The integration of chromatin labeling with live cell imaging can directly observe the dynamics of the specific chromatin in a nucleus.…”