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AssigneeNew England Biolabs, Inc.
InventorsGregory Lohman, Vladimir Potapov, John M. Pryor, Rebecca Kucera, Katharina Bilotti, Richard D. Morgan
Estimating the term…
Ordered assembly of large numbers of fragments into a single large DNA have been improved in both frequency and fidelity of the assembled product. This has been achieved by novel compositions and methods that are utilized in a computer system that integrates comprehensive ligation data from multiple sources to provide optimized synthetic overhangs or overhangs from restriction endonuclease cleavage on DNA fragments for assembly by ligation. Intragenic cut sites are avoided by the use of a novel restriction endonuclease which recognizes 7 nucleotides (bases) and cuts DNA to create 4-base overhangs with the help of a synthetic activator oligonucleotide. Variations in ligation preferences by different ligases provide extra precision in assembly reactions. The use of the improved methods are exemplified by the successful assembly from 52 fragments of a viral genome and also a 52 fragment ordered assembly of a bacteria operon.
A synthetic self-complementary oligonucleotide that comprises a double-stranded region and a single strand loop, wherein the double-stranded region contains a recognition sequence for PaqCI, has unligatable 3′ and 5′ ends and cannot be cleaved by PaqCI.
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