Four fluor sequencing

NEW YORK—In trying to reach the $1000 genome, sequencing-by-synthesis (SBS) methods have gained in popularity; however, like most sequencing experiments, SBS requires scientists to perform four separate reactions for each of the nucleotides. Recently, researchers at Columbia University have addressed this problem by developing fluorescently tagged nucleotides that allow them to sequence DNA in a single reaction.

 

As reported in PNAS, the researchers synthesized four terminator nucleotide analogues modified at the 3′ OH with an allyl blocking group and at the 5-position (pyrimidines) or 7-position (purines) with allyl-fluorophore tags based on Bodipy or rhodamine. Because each tag has distinct absorption and emission spectra, the four nucleotides can be run in the same reaction. The analogues were also designed so that the blocking group and tags can be chemically cleaved using a palladium catalyst following each synthesis and detection step.

 

The researchers showed that DNA polymerase could incorporate the four terminator nucleotides into nascent DNA chains with high fidelity. Furthermore, they found the tagged method worked better than pyrosequencing (another SBS method) at correctly sequencing homopolymeric stretches (e.g., polyA sequences). The next step in method development, say the researchers, will be to optimize reaction conditions to extend the sequence reads from 20 nucleotides to lengths more comparable to pyrosequencing (100 nucleotides) or perhaps even Sanger sequencing (800 nucleotides).