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Novel method of cell-free in vitro synthesis of the human fibroblast growth factor 1 gene.

Zuo P, Rabie AB - J. Biomed. Biotechnol. (2010)

Bottom Line: The new method involved two steps: (1) the design of the DNA oligonucleotides to be assembled and (2) the assembly of multiple oligonucleotides by PCR to generate the whole FGF1 gene.The procedure lasted a total of only 2 days, compared with 2 weeks for the conventional procedure.This method of gene synthesis is expected to facilitate various kinds of complex genetic engineering projects that require rapid gene amplification, such as cell-free whole-DNA library construction, as well as the construction of new genes or genes that contain any mutation, restriction site, or DNA tag.

View Article: PubMed Central - PubMed

Affiliation: Orthodontics, Faculty of Dentistry, The University of Hong Kong, 2/F, Prince Philip Dental Hospital, 34 SAR, Hong Kong. pzuo@hkucc.hku.hk

ABSTRACT
Recombinant DNA projects generally involve cell-based gene cloning. However, because template DNA is not always readily available, in vitro chemical synthesis of complete genes from DNA oligonucleotides is becoming the preferred method for cloning. This article describes a new, rapid procedure based on Taq polymerase for the precise assembly of DNA oligonucleotides to yield the complete human fibroblast growth factor 1 (FGF1) gene, which is 468 bp long and has a G+C content of 51.5%. The new method involved two steps: (1) the design of the DNA oligonucleotides to be assembled and (2) the assembly of multiple oligonucleotides by PCR to generate the whole FGF1 gene. The procedure lasted a total of only 2 days, compared with 2 weeks for the conventional procedure. This method of gene synthesis is expected to facilitate various kinds of complex genetic engineering projects that require rapid gene amplification, such as cell-free whole-DNA library construction, as well as the construction of new genes or genes that contain any mutation, restriction site, or DNA tag.

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Confirmation of assembled plasmid containing the FGF1 gene. The pcDNA3.1/V5-His-TOPO-FGF1 plasmid was cleaved by HindIII and XhoI. Lane 1: DNA molecular weight markers. Lane 2: Two digestion fragments of 5433 bp and 561 bp.
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fig3: Confirmation of assembled plasmid containing the FGF1 gene. The pcDNA3.1/V5-His-TOPO-FGF1 plasmid was cleaved by HindIII and XhoI. Lane 1: DNA molecular weight markers. Lane 2: Two digestion fragments of 5433 bp and 561 bp.

Mentions: After the crude PCR reaction mixtures that contained the product were directly cloned into the cloning vector, we readily obtained clones that contained the correct sequences. Double digestion of extracted plasmid DNA with HindIII and XhoI yielded two fragments on a 1% agarose gel, of sizes 5433 bp and 561 bp, which represented the linear vector pcDNA3.1/V5-His-TOPO and the FGF1 gene, respectively (Figure 3). Because the fragment yielded by HindIII and XhoI digestion that contained the gene also contained some base pairs from the vector, it was 31 bp longer than the corresponding earlier PCR fragment. The DNA sequence of the FGF1 gene in the complete pcDNA3.1/V5-His-TOPO-FGF1 construct was 100% in agreement with the sequences from the NCBI gene bank (data not shown).


Novel method of cell-free in vitro synthesis of the human fibroblast growth factor 1 gene.

Zuo P, Rabie AB - J. Biomed. Biotechnol. (2010)

Confirmation of assembled plasmid containing the FGF1 gene. The pcDNA3.1/V5-His-TOPO-FGF1 plasmid was cleaved by HindIII and XhoI. Lane 1: DNA molecular weight markers. Lane 2: Two digestion fragments of 5433 bp and 561 bp.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2913909&req=5

fig3: Confirmation of assembled plasmid containing the FGF1 gene. The pcDNA3.1/V5-His-TOPO-FGF1 plasmid was cleaved by HindIII and XhoI. Lane 1: DNA molecular weight markers. Lane 2: Two digestion fragments of 5433 bp and 561 bp.
Mentions: After the crude PCR reaction mixtures that contained the product were directly cloned into the cloning vector, we readily obtained clones that contained the correct sequences. Double digestion of extracted plasmid DNA with HindIII and XhoI yielded two fragments on a 1% agarose gel, of sizes 5433 bp and 561 bp, which represented the linear vector pcDNA3.1/V5-His-TOPO and the FGF1 gene, respectively (Figure 3). Because the fragment yielded by HindIII and XhoI digestion that contained the gene also contained some base pairs from the vector, it was 31 bp longer than the corresponding earlier PCR fragment. The DNA sequence of the FGF1 gene in the complete pcDNA3.1/V5-His-TOPO-FGF1 construct was 100% in agreement with the sequences from the NCBI gene bank (data not shown).

Bottom Line: The new method involved two steps: (1) the design of the DNA oligonucleotides to be assembled and (2) the assembly of multiple oligonucleotides by PCR to generate the whole FGF1 gene.The procedure lasted a total of only 2 days, compared with 2 weeks for the conventional procedure.This method of gene synthesis is expected to facilitate various kinds of complex genetic engineering projects that require rapid gene amplification, such as cell-free whole-DNA library construction, as well as the construction of new genes or genes that contain any mutation, restriction site, or DNA tag.

View Article: PubMed Central - PubMed

Affiliation: Orthodontics, Faculty of Dentistry, The University of Hong Kong, 2/F, Prince Philip Dental Hospital, 34 SAR, Hong Kong. pzuo@hkucc.hku.hk

ABSTRACT
Recombinant DNA projects generally involve cell-based gene cloning. However, because template DNA is not always readily available, in vitro chemical synthesis of complete genes from DNA oligonucleotides is becoming the preferred method for cloning. This article describes a new, rapid procedure based on Taq polymerase for the precise assembly of DNA oligonucleotides to yield the complete human fibroblast growth factor 1 (FGF1) gene, which is 468 bp long and has a G+C content of 51.5%. The new method involved two steps: (1) the design of the DNA oligonucleotides to be assembled and (2) the assembly of multiple oligonucleotides by PCR to generate the whole FGF1 gene. The procedure lasted a total of only 2 days, compared with 2 weeks for the conventional procedure. This method of gene synthesis is expected to facilitate various kinds of complex genetic engineering projects that require rapid gene amplification, such as cell-free whole-DNA library construction, as well as the construction of new genes or genes that contain any mutation, restriction site, or DNA tag.

Show MeSH