Long multiple fusion using a Pfu polymerase mix successfully assembled linear DNA constructs up to 20 kb with an accuracy of at least one error per 6.6 kb.
The long multiple fusion method allows for precise, simultaneous assembly of multiple DNA fragments up to 20 kb, facilitating complex genetic engineering projects.
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.
Nikolai A Shevchuk (Wed,) reported a other. Long multiple fusion (long PCR-based fusion) was evaluated on Assembly of linear DNA constructs up to 20 kb. Long multiple fusion using a Pfu polymerase mix successfully assembled linear DNA constructs up to 20 kb with an accuracy of at least one error per 6.6 kb.
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