So the ACS conference is no more. Actually, it feels like it’s been winding down since Tuesday, what with all the big banners saying “See you at the next meeting”, the posters advertising taxi services to the airport, and people wandering around with their luggage. I think it’s a shame that so many people have already gone home by the last day, especially for those who are still presenting. They may not be the biggest names, but it doesn’t seem fair that their potential audience is reduced before they even start.

As I mentioned before, this was my first ACS, and I think it’s lived up to expectations. I’ve enjoyed the chemistry (of course) but also the people watching. It seems that more senior chemists are predisposed towards facial hair (especially splendid moustaches), whereas the younger crowd are rebelling by generally being clean-shaved (although a trendy goatee beard is permitted).

Technology now allows for some mind-blowing multi-media presentations, but also inevitably to a random scattering of pop-up error messages. Still, I’ve enjoyed some of the visual humour. My favourite was the speaker who, when discussing the various organisms that have had their genomes sequenced, provided an image of each species. There was a picture of fruit-fly, a worm, and a chimpanzee. And right next to the chimp, representing humanity, there was George Bush. Say no more.

Anyway, I’ll be flying back home tomorrow (oh joy, another 8 hour flight with nothing to do but watch films about penguins. Why is it always penguins?), and then I’m going to lie down in a darkened room for a week. Really.

Andy

Andrew Mitchinson (Associate Editor, Nature)

Two recent studies report on complementary methodologies to assemble long DNA segments.

1. Homme Hellinga and colleagues describe the so-called “protein fabrication automation” (PFA) pipeline to synthesize de novo open-reading frames from oligonucleotides (Cox et al, 2007). The method involves a robust two-step PCR assembly scheme (performed with the KOD polymerase) followed by a genetic selection step:
   • First, an inside-out PCR (ION–PCR) with 5-6 overlapping 50bp oligos is performed to generate 400bp fragments
   • Second, a splice overlap extension PCR (SOE–PCR) is run to assemble the fragments into a complete ORF
   • Third, frameshift mutations supressed by genetic selection using an in-frame C-terminal fusion with chloramphenicol acetyl transferase

   Automation allows synthesizing up to 48 ORFs (1.3 kb in length) within 2 weeks.

2. In the second paper, Stephen Elledge and coworkers describe the “sequence and ligation-idependent cloning” (SLIC) method (Li and Elledge, 2007). In this approach, the T4 DNA polymerase is used to produce (in absence of nucleotides) 5’ overhangs within 30bp-long homology regions. Equimolar mix of the fragments and vector to be assembled are incubated in presence of RecA and ATP and transformed into bacteria to complete repair and generate recombinant DNA. Using this strategy, the authors report efficient three-way and five-way assembly reactions involving 300-400bp PCR fragments and even a successful ten-way assembly, albeit with much reduced efficiency (20% or correct clones).

While the first method is primarily designed for the automated high-throughput generation of coding sequences, the second is a general and flexible method for the assembly of various types of genetic elements including coding, regulatory, etc sequences. One could perhaps dream of integrating both methods to achieve super-long and/or combinatorial DNA synthesis?