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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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Oligonucleotides used to synthesize FGF1.  A total of 14 oligonucleotides, 50–70 nucleotides in length, collectively encoded both strands of the FGF1 gene.
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fig1: Oligonucleotides used to synthesize FGF1. A total of 14 oligonucleotides, 50–70 nucleotides in length, collectively encoded both strands of the FGF1 gene.

Mentions: We designed and ordered 14 oligonucleotides, ranging from 50 to 70 nucleotides in length, which collectively encoded both strands of the FGF1 gene (Table 1 and Figure 1). The sense and antisense strands each consisted of 7 oligonucleotides. Complementary oligonucleotides were expected to overlap when the melting temperature (Tm) exceeds 60°C (Figure 1). We incorporated bolded sequences that overlapped with the parts of the sequence of the plasmid pcDNA3.1/V5-His-TOPO (Invitrogen, Carlsbad) to facilitate cloning into this plasmid [13]. Oligonucleotides and all reagents unless otherwise stated were purchased from Sigma-Aldrich (St Louis, MO, USA).


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

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

Oligonucleotides used to synthesize FGF1.  A total of 14 oligonucleotides, 50–70 nucleotides in length, collectively encoded both strands of the FGF1 gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Oligonucleotides used to synthesize FGF1. A total of 14 oligonucleotides, 50–70 nucleotides in length, collectively encoded both strands of the FGF1 gene.
Mentions: We designed and ordered 14 oligonucleotides, ranging from 50 to 70 nucleotides in length, which collectively encoded both strands of the FGF1 gene (Table 1 and Figure 1). The sense and antisense strands each consisted of 7 oligonucleotides. Complementary oligonucleotides were expected to overlap when the melting temperature (Tm) exceeds 60°C (Figure 1). We incorporated bolded sequences that overlapped with the parts of the sequence of the plasmid pcDNA3.1/V5-His-TOPO (Invitrogen, Carlsbad) to facilitate cloning into this plasmid [13]. Oligonucleotides and all reagents unless otherwise stated were purchased from Sigma-Aldrich (St Louis, MO, USA).

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