期刊
BIOTECHNOLOGY LETTERS
卷 34, 期 4, 页码 721-728出版社
SPRINGER
DOI: 10.1007/s10529-011-0832-0
关键词
Catenin beta 1 gene; Gene synthesis; Ligation; Oligodeoxynucleotide; Polymerase chain reaction
Synthesis of long DNA fragments is often associated with mutations and requires multiple DNA manipulation steps. A novel DNA synthesis method, referred to as patch oligodeoxynucleotide synthesis (POS) to assembly long DNA fragments is presented here. This method involves connection of two types of oligodeoxynucleotides: long constructional oligonucleotides (COs) and short patch oligonucleotides (POs). Long COs were connected by a ligase with the aid of POs, which were complementary to both adjacent COs to help remove secondary structures during assembly. The partial double-stranded DNA template that was formed was then amplified by PCR. Accordingly, we synthesized SV40 polyadenylation signal sequences (187 bp), a codon-optimized yellow fluorescent protein gene (678 bp), and Rattus norvegicus catenin beta 1 (2,352 bp). This presented method can be broadly applied to synthesize DNA fragments of varying lengths with great convenience.
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