A facile method for the construction of synthetic genes (original) (raw)
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Artificial Gene Synthesis in Vitro
Gene of interest is rare in nature, or the genetic material containing such genes is difficult to obtain or isolate, de nova chemical synthesis of complete genes is a preferred method of cloning. One can now envisage synthesizing not only a new gene, but also a whole library of genes or mutants of a single gene for bioengineering applications, structural studies, drug development, and combinatorial biology and so on. The major technological driving force of this field is to make increasingly long stretches of error free DNA at reduced cost and also on improving the design of genes for specific purposes. This can be achieved by synthesizing oligos with overlapping ends, then annealing these oligos and extending to form a full-length gene. But due to the inherent nature of oligo synthesis, mutations, especially deletions occur frequently during the synthesis process. This situation dramatically limits the gene size and increase the cost of gene synthesis. To overcome this problem, a novel gene synthesis platform has been developed to efficiently synthesize the gene of larger size with no mutation. The synthetic gene can be optimized for expression and constructed for easy mutational manipulation without regard to the parent genome. Our program requires simple input information i.e. amino acid sequence of the target protein and melting temperature (needed for the gene assembly) of synthetic oligonucleotides. The program output a series of oligonucleotides sequences with codon optimized for expression in an organism of choice. Those oligonucleotides are characterized by highly homogenous melting temperatures and a minimize tendency for hairpin formation. With the help of this program and a two step PCR method, synthetic genes more than 1000bp can constructed. The approach present here simplifies the production of proteins from a wide variety of organisms for genomics-based studies.
A new method for the synthesis of a structural gene
Nucleic Acids Research, 1990
A novel method of synthesizing a structural gene or gene fragment, consisting of the first synthesis of a single-stranded DNA (ssDNA), has been developed. As a preliminary test of this method, four synthetic genes or gene fragments have been synthesized. The first one with 396 base pairs (b.p.) codes for the mature rbcS from wheat, the next two with 370 and 342 b.p. respectively, for two half molecules of a gene for trichosanthin and the last one with 315 b.p. for the Nterminal 1-102 residues of human prourokinase. In all these syntheses, a plus-stranded DNA of the target gene was generally assembled by a stepwise or one step T4 DNA ligase reaction of six oligonucleotides (A, *pB, *pC, *pD, *pE and *pF) of 30-71 nucleotides long in the presence of two terminal complementary oligonucleotides (Ab' and eF') and three short interfragment complementary oligonucleotides (bc, cd and de). After purification, the synthetic ssDNA was inserted into a cloning vector, pWR13. The resulting product was directly used to transform a host cell. The structure of the cloned synthetic gene was confirmed by DNA sequence analysis.
DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis
Nucleic Acids Research, 2002
The availability of sequences of entire genomes has dramatically increased the number of protein targets, many of which will need to be overexpressed in cells other than the original source of DNA. Gene synthesis often provides a fast and economically efficient approach. The synthetic gene can be optimized for expression and constructed for easy mutational manipulation without regard to the parent genome. Yet design and construction of synthetic genes, especially those coding for large proteins, can be a slow, difficult and confusing process. We have written a computer program that automates the design of oligonucleotides for gene synthesis. Our program requires simple input information, i.e. amino acid sequence of the target protein and melting temperature (needed for the gene assembly) of synthetic oligonucleotides. The program outputs a series of oligonucleotide sequences with codons optimized for expression in an organism of choice. Those oligonucleotides are characterized by highly homogeneous melting temperatures and a minimized tendency for hairpin formation. With the help of this program and a two-step PCR method, we have successfully constructed numerous synthetic genes, ranging from 139 to 1042 bp. The approach presented here simplifies the production of proteins from a wide variety of organisms for genomics-based studies.
Journal of Microbiological Methods, 2009
Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primermediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA-PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers.
Beilstein Journal of Organic Chemistry, 2014
Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide "letters", the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson-Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so.
Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides
Gene, 1995
Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling [Stemmer, Nature 370 (1994a) 389-391], does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 [3-1actamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (ntl in length. The synthetic gene was PCR amplified and cloned in a vector containing the tetracycline-resistance gene (Tc R) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the Tc R colonies were Ap R, making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the s-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.
A Rapid PCR Based Method for Gene Synthesistrol
Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for synthesis of long segments of DNA. The method involves two steps: 1) PCR assembling of chemically synthezyed DNA oligomers and 2) specific amplification of the full length DNA sequence . Compared with the previously published methods, the this method is rapid (2 days) and suitable for synthesizing long segments of DNA (2 kb) with high G + C contents, repetitive sequences or complex secondary structures.
Oligonucleotide synthesis using the manual phosphotriester method
Methods in molecular biology (Clifton, N.J.), 1988
During the past few years, synthetic DNA, used as a primer in DNA polymerization, in site-directed mutagenesis or as a probe in gene selection, has assumed a central role in recombinant DNA technology (1). This was made possible by the development of methods for efficient solid phase chemical synthesis. Deoxyribonucleotides are ideally suited to solid-phase synthesis since their relative chemical uniformity allows for application of oligodeoxyribonucleotide (oligonucleotide) purification techniques, which are largely dependent on chain length. Hence, the potential disadvantage of omitting purification after each coupling step is minimized. This contrasts with peptide synthesis in which purification of the final product is more difficult, thereby placing greater demands on coupling efficiency. In practice, coupling efficiencies of at least 95% are now attainable in oligonucleotide synthesis because of the availability of highly reactive mononucleotides and specially developed couplin...