[Assembly of apoptin gene using oligodeoxyribonucleotides in vitro].
OBJECTIVE
To explore the method for in vitro gene assembly of apoptin-encoding DNA sequence, for instance, using a large number of oligodeoxyribonucleotides (oligos).


METHODS
Based on the encoding sequence of apoptin gene (GeneBank accession number AY171617), a number of oligos were designed to assembly apoptin gene in pfu mix reaction system, and each oligo was 40 nucleotides (nt) in length, in which synonymous codon substitution was used to eliminate the restriction enzyme sites of Bgl II ( position 172, agatct-agatcc) and Hind III (position 306, aagctt-aatcct). The assembly mixture was further diluted and amplified with two end oligos. The targeting sequence was gel-purified, amplified for one more time, followed by the addition of T to the 3' end in the presence of Taq polymerase and dATP before cloning into pGEM-T easy vector. The positive clones were confirmed by restriction enzyme digestion and sequence analysis.


RESULTS
The synthetic mixture presented obvious "tails" in the first PCR for assembly. After dilution of the mixture and amplification with two end oligos, clear DNA ladder bands with a clear targeted band were yielded. After PCR, the targeted gene was cloned into pGEM-T easy vector, and the positive clones were confirmed by sequence analysis with be identical to the designed coding sequence of apoptin gene.


CONCLUSIONS
Gene assembly is a rapid and cost-effective approach for synthesis of genes or vectors, which allows simultaneous mutagenesis of several genes in vitro.
Chen
Xue-Qing
XQ
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. chenxq@bfbt.com
Wang
Ya-Dong
YD
Sun
Yong
Y
Duan
You-Cai
YC
Ma
Gao-Feng
GF
D001483:Base Sequence; D036022:Capsid Proteins; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction
D036022:Capsid Proteins; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; C091717:VP3 protein, Chicken anemia virus