Life only exists because of the ability of cells to replicate, and death can come early when a cell’s replication spins out of control. Yet exactly where eukaryotic cells start when their large genomes need to be copied is still somewhat of a mystery. Studies looking into replication origins have largely focused on simple organisms with smaller genomes. Observing this process in large genomes like ours is difficult because eukaryotic cells have up to 50,000 replication start points per cell per cycle, and even the most commonly observed replication origin in the genome functions as such in just 10 percent of cells. To find answers to the question of where and when replication starts in individual cells, one needs a technology that can image and identify large single DNA molecules that are fluorescently labeled at high resolution and throughput… and of course we knew just what technology that could be!
A team led by Dr. Nick Rhind at the University of Massachusetts Medical School worked with some of Bionano’s brightest minds to develop a method that can visualize these replication origins on Saphyr. The protocol is surprisingly simple: transfect synchronized and arrested HeLa cells with what’s basically Bionano’s red labeling mix (consisting of fluorescently labeled nucleotides), allow the cell cycle to resume and prepare DNA using Bionano’s standard workflow. Use our standard NLRS labeling kit with a nicking endonuclease to label the molecules green, and image on Saphyr. The green signal is used to assemble and align the molecules to the reference, the red signal shows where on those molecules the replication originated.
The resulting images (like the one shown here) are stunning and the 290x coverage of the genome allowed the team to identify early-firing human replication origins that occur in as few as 1% of cells.
Replication mapping is not an application that’s currently supported by Bionano, so if you would like to find out how to run your own such experiments on Saphyr, read the paper posted on bioRxiv here: Genome-Wide Identification of Early-Firing Human Replication Origins by Optical Replication Mapping
