| PCR-based accurate synthesis of long DNA sequences. | |
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MedLine Citation:
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PMID: 17406309 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Here we describe a simple and rapid method for assembly and PCR-based accurate synthesis (PAS) of long DNA sequences. The PAS protocol involves the following five steps: (i) design of the DNA sequence to be synthesized and of 60-bp overlapping oligonucleotides to cover the entire DNA sequence; (ii) purification of the oligonucleotides by PAGE; (iii) first PCR, to synthesize DNA fragments of 400-500 bp in length using 10 inner (template) and two outer (primer) oligonucleotides; (iv) second PCR, to assemble the products of the first PCR into the full-length DNA sequence; and (v) cloning and verification of the synthetic DNA by sequencing and, if needed, error correction using an overlap-extension PCR technique. This method, which takes approximately 1 wk, is suitable for synthesizing diverse types of long DNA molecule. We have successfully synthesized DNA fragments from 0.5 to 12.0 kb, with high G+C content, repetitive sequences or complex secondary structures. The PAS protocol therefore provides a simple, rapid, reliable and relatively inexpensive method for synthesizing long, accurate DNA sequences. |
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Authors:
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Ai-Sheng Xiong; Quan-Hong Yao; Ri-He Peng; Hui Duan; Xian Li; Hui-Qin Fan; Zong-Ming Cheng; Yi Li |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Nature protocols Volume: 1 ISSN: 1750-2799 ISO Abbreviation: Nat Protoc Publication Date: 2006 |
Date Detail:
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Created Date: 2007-04-04 Completed Date: 2007-09-21 Revised Date: 2008-03-24 |
Medline Journal Info:
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Nlm Unique ID: 101284307 Medline TA: Nat Protoc Country: England |
Other Details:
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Languages: eng Pagination: 791-7 Citation Subset: IM |
Affiliation:
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Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai 201106, The People's Republic of China. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Base Composition Base Sequence DNA / chemical synthesis*, genetics* Genes, Synthetic / genetics Mutation Polymerase Chain Reaction / methods* |
| Chemical | |
Reg. No./Substance:
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9007-49-2/DNA |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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