A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.
These results compare favorably with those reported in earlier periods. With programs such as the IRP, patients with short bowel secondary to intestinal atresia can show improvement in liver function and nutritional parameters, and discontinue PN, avoiding the need for transplantation.
From audacious space missions and quantum physics, to clean cookstoves and coral nurseries, this year's finalists for the AAAS/Suburu SB&F Prizes for Excellence in Science Books dare to go where few children's titles have gone before. Sponsored by Subaru of America and facilitated by the American Association for the Advancement of Science (AAAS, the publisher of
Science
), the competition celebrates high-quality children's science books. Read on for reviews written by the staff of the
Science
family of journals and a few friends.
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