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French Researchers Use Novel Approach to Sequence L. Lactis Genome

NEW YORK, April 16 - French scientists have sequenced the genome of Lactococcus lactis , a bacterium that plays an important role in the manufacturing of cheese, using a novel two-step sequencing strategy, the researchers reported in Monday's issue of Genome Research .

To sequence the L. lactis genome, Alexei Sorokin, a genomics researcher at the National Institute of Agronomic Research in Jouy en Josas, France, and his colleagues at the National Sequencing Center in Evry, France, employed a sequencing strategy that requires three to four-fold fewer sequencing reactions that the standard whole genome "shotgun" method. 

In the first step, called diagnostic genome sequencing, the researchers cloned relatively short genome fragments using E. coli vectors and sequenced a limited number of randomly-chosen clones. The team then employed a novel PCR technique, called multiplex long accurate PCR, to connect the resulting contigs.

In a second step, the researchers randomly sequenced additional clones, followed by primer walking on PCR-generated templates, to verify the accuracy of the sequence.

"We designated [this second step] 'shotgun polishing' and concluded that the strategy presented here can be a good alternative to the fully random strategy used in most cases," wrote the authors in the paper. "Its advantages should increase even more when a greater number of completely sequenced and thoroughly annoted bacterial genomes becomes available."

With about 2.4 million base pairs encoding for 2,310 proteins, the L. lactis  genome is not excessively large--it's about one thousandth the size of the human genome--but understanding its sequence may lead to improvements in the production of cheese, as well as advances in understanding the organism's medically relevant cousins, the genus Streptococcus, scientists say.

"This organism is critically important to the dairy industry," said Marlene Belfort, a geneticist at the New York State Department of Health who has studied L. lactis . "There's a huge amount to be learned in terms of the mechanisms of various processes."
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