by Aaron J. Sender
A DNA engineering method provides ample amplification
“We didn’t set out to get around PCR,” says Gene Bridges chairman Francis Stewart about the origins of Red/ET Recombination, which he invented at EMBL. “We set out to find a way to engineer BACs.”
It just so happened that the DNA engineering technique, whose patent was recently issued, is also an efficient and precise way to amplify specific genomic DNA sequences in vivo. “It’s an alternative to PCR,” says Stewart.
To modify the genetic sequence of BACs, researchers are limited by the inability to find appropriate restriction sites. They take out the piece they want, insert it into another vector, reinsert it into the BAC, and screen for recombinants — a long and laborious process that could take months. Red/ET simplifies the process. It uses homologous recombination to introduce point mutations or very large pieces of DNA directly into the clone, by mimicking the way a lambda phage transforms E. coli into a host for its genetic material.
Red/ET may also be used to clone a region directly out of a mammalian genome in preparation for analysis, such as sequencing, while eliminating some limitations of PCR. “It has a couple of advantages over PCR,” says Stewart. One is that PCR tends to introduce errors as it makes copies. “Red/ET is a very high-fidelity process,” he says. “Because the replication is done by the endogenous E. coli machinery, it’s fully proofread. It’s not a sloppy in vitro process.”
Stewart’s process can also handle larger pieces of DNA. “Whereas PCR gets quite ugly as you try to get above five or six kilobases, in our process it’s simple to pick up pieces that are 50 kilobases,” he says.
Where Red/ET falls short is sensitivity. “With PCR you can take a single cell, put in a PCR reaction and you get your robust result. With this methodology, as it stands, we can’t do that,” he says. “We need at least several micrograms of genomic DNA.”
Gene Bridges, based in Dresden, Germany, is now seeking commercial licensees for the technology. It also offers DNA engineering services and DIY kits for academics.