PCR-based accurate synthesis of long DNA sequences

Here we describe a simple and rapid method for assembly and PCR-based accurate synthesis (PAS) of long DNA sequences. The PAS protocol involves the following five steps: (i) design of the DNA sequence to be synthesized and of 60-bp overlapping oligonucleotides to cover the entire DNA sequence; (ii) purification of the oligonucleotides by PAGE; (iii) first PCR, to synthesize DNA fragments of 400-500 bp in length using 10 inner (template) and two outer (primer) oligonucleotides; (iv) second PCR, to assemble the products of the first PCR into the full-length DNA sequence; and (v) cloning and verification of the synthetic DNA by sequencing and, if needed, error correction using an overlap-extension PCR technique. This method, which takes approximately 1 wk, is suitable for synthesizing diverse types of long DNA molecule. We have successfully synthesized DNA fragments from 0.5 to 12.0 kb, with high G+C content, repetitive sequences or complex secondary structures. The PAS protocol therefore provides a simple, rapid, reliable and relatively inexpensive method for synthesizing long, accurate DNA sequences.     

A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences

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 (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are ~500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5–7 days) and suitable for synthesizing long segments of DNA (5–6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.     

Determination of DNA sequences essential for FLP-mediated recombination by a novel method

The yeast 2-micron circle plasmid encodes a protein, FLP, that mediates site-specific recombination across the two FLP-binding sites of the plasmid. We have used a novel technique, "exonuclease-treated substrate analysis," to determine the minimal duplex DNA sequence needed for this recombination event. A linear DNA containing two FLP sites in a direct orientation was treated with the double-strand specific 3'-exonuclease, exonuclease III, to generate molecules with a nested set of single-strand deletions that extended into one of the FLP sites. The DNA was then end-labeled at the sites of the deletions and used as a substrate for recombination in vitro. FLP-mediated recombination between two FLP sites excised a restriction endonuclease cleavage site from the DNA. Comparison of the fragments produced by restriction enzyme digestion of untreated and FLP-treated DNA showed to the nucleotide the duplex DNA sequence required for FLP-mediated recombination. To examine essential sequences in the opposite DNA strand, similar experiments were done using the 5'-exonuclease encoded by phage T7. The minimal essential duplex DNA sequence lies within the region of the FLP site that was previously shown to be protected from nuclease digestion in the presence of FLP. A modified form of this technique can be used to study the minimal sequence requirements of site-specific DNA binding proteins.     

Signal-exon trap: a novel method for the identification of signal sequences from genomic DNA

We describe a genomic DNA-based signal sequence trap method, signal-exon trap (SET), for the identification of genes encoding secreted and membrane-bound proteins. SET is based on the coupling of an exon trap to the translation of captured exons, which allows screening of the exon-encoded polypeptides for signal peptide function. Since most signal sequences are expected to be located in the 5′-terminal exons of genes, we first demonstrate that trapping of these exons is feasible. To test the applicability of SET for the screening of complex genomic DNA, we evaluated two critical features of the method. Specificity was assessed by the analysis of random genomic DNA and efficiency was demonstrated by screening a 425 kb YAC known to contain the genes of four secretory or membrane-bound proteins. All trapped clones contained a translation initiation signal followed by a hydrophobic stretch of amino acids representing either a known signal peptide, transmembrane domain or novel sequence. Our results suggest that SET is a potentially useful method for the isolation of signal sequence-containing genes and may find application in the discovery of novel members of known secretory gene clusters, as well as in other positional cloning approaches.     

HYLAS: program for generating H curves (abstract three-dimensional representations of long DNA sequences)

The computer program HYLAS generates from a standard DNA lettersequence a three-dimensional space curve (H curve) which embodiesthe entire information content of the original nucleotide sequence.The program can display H curves either as two-dimensional (frontand side view) projections or as stereo-pair images. The curvescan be marked at specific nucleotide locations, annotated, rotatedfor observation from any viewing angle, and manipulated forconvenient side-by-side comparisons. Unlike the cumbersome lettersequences, H curves can be drastically condensed in size withoutlosing their ability to reflect the global nucleotide-distributionpattern of the entire DNA sequence. Often, biologically importantloci can be visually identified on the H curves. HYLAS is writtenin FORTRAN with separate mainframe (IBM- VM/CMS) and microcomputer(MS-DOS) versions. It uses the Tektronix-TCS library of graphicsubroutines. Received on October 24, 1988; accepted on July 15, 1989     

Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences

A novel thermodynamically-balanced inside-out (TBIO) method of primer design was developed and compared with a thermodynamically-balanced conventional (TBC) method of primer design for PCR-based gene synthesis of codon-optimized gene sequences for the human protein kinase B-2 (PKB2; 1494 bp), p70 ribosomal S6 subunit protein kinase-1 (S6K1; 1622 bp) and phosphoinositide-dependent protein kinase-1 (PDK1; 1712 bp). Each of the 60mer TBIO primers coded for identical nucleotide regions that the 60mer TBC primers covered, except that half of the TBIO primers were reverse complement sequences. In addition, the TBIO and TBC primers contained identical regions of temperature- optimized primer overlaps. The TBC method was optimized to generate sequential overlapping fragments (~0.4–0.5 kb) for each of the gene sequences, and simultaneous and sequential combinations of overlapping fragments were tested for their ability to be assembled under an array of PCR conditions. However, no fully synthesized gene sequences could be obtained by this approach. In contrast, the TBIO method generated an initial central fragment (~0.4–0.5 kb), which could be gel purified and used for further inside-out bidirectional elongation by additional increments of 0.4–0.5 kb. By using the newly developed TBIO method of PCR-based gene synthesis, error-free synthetic genes for the human protein kinases PKB2, S6K1 and PDK1 were obtained with little or no corrective mutagenesis.     

Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis

A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.     

Nucleotide sequences of tobacco chloroplast genes for elongator tRNAMet and tRNAVal (UAC): the tRNAVal (UAC) gene contains a long intron.

The nucleotide sequences of tobacco chloroplast genes for elongator tRNAMet and tRNAVal (UAC) have been determined. The tRNAVal gene contains a 571 base pairs intron located in the anticodon loop. The tRNAVal gene is transcribed as a 750 bases precursor RNA molecule. Both tRNAs deduced from the DNA sequences show 97% sequence homologies with those of spinach chloroplasts.     

A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase

A simple and rapid method for determining nucleotide sequences in single-stranded DNA by primed synthesis with DNA polymerase is described. It depends on the use of Escherichia coli DNA polymerase I and DNA polymerase from bacteriophage T4 under conditions of different limiting nucleoside triphosphates and concurrent fractionation of the products according to size by ionophoresis on acrylamide gels. The method was used to determine two sequences in bacteriophage φX174 DNA using the synthetic decanucleotide A-G-A-A-A-T-A-A-A-A and a restriction enzyme digestion product as primers.     

ROKU: a novel method for identification of tissue-specific genes

Background  

One of the important goals of microarray research is the identification of genes whose expression is considerably higher or lower in some tissues than in others. We would like to have ways of identifying such tissue-specific genes.     

Extrachromosomal DNA of pea-root (Pisum sativum) has repeated sequences and ribosomal genes

Summary Restriction endonuclease digestion and Southern blotting procedure were used to determine differences between extrachromosomal, nuclear, plastid, and mitochondrial DNAs from meristematic cells of cultured pea roots.Extrachromosomal and nuclear DNA are highly methylated and neither DNA is homologous to plastid or mitochondrial DNA. Hybridization of extrachromosomal DNA to nuclear DNA indicated that extrachromosomal DNA differed quantitatively from total nuclear DNA in repetitive sequences. Cloned rDNA showed that extrachromosomal DNA contains rRNA genes but the hybridization signal indicated that the copy number was less than that expected if the molecules were amplified. These and cytological findings suggest that extrachromosomal DNA is involved in or a product of genomic changes associated with the onset of differentiation by precursor cells of vascular parenchyma and the root cap.     

SeeDNA： A Visualization Tool for K-string Content of Long DNA Sequences and Their Randomized Counterparts

An interactive tool to visualize the K-string composition of long DNA sequences including bacterial complete genomes is described. It is especially useful for exploring short palindromic structures in the sequences. The SeeDNA program runs on Red Hat Linux with GTK support. It displays two-dimensionai (2D) or one-dimensional (1D) histograms of the K-string distribution of a given sequence and/or its randomized counterpart. It is also capable of showing the difference of K-string distributions between two sequences. The C source code using the GTK package is freely available.     

Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes

To investigate the feasibility of conducting a genomic-scale protein labeling and localization study in Escherichia coli, a representative subset of 23 coding DNA sequences (CDSs) was selected for chromosomal tagging with one or more fluorescent protein genes (EGFP, EYFP, mRFP1, DsRed2). We used λ-Red recombination to precisely and efficiently position PCR-generated DNA targeting cassettes containing a fluorescent protein gene and an antibiotic resistance marker, at the C-termini of the CDSs of interest, creating in-frame fusions under the control of their native promoters. We incorporated cre/loxP and flpe/frt technology to enable multiple rounds of chromosomal tagging events to be performed sequentially with minimal disruption to the target locus, thus allowing sets of proteins to be co-localized within the cell. The visualization of labeled proteins in live E. coli cells using fluorescence microscopy revealed a striking variety of distributions including: membrane and nucleoid association, polar foci and diffuse cytoplasmic localization. Fifty of the fifty-two independent targeting experiments performed were successful, and 21 of the 23 selected CDSs could be fluorescently visualized. Our results show that E. coli has an organized and dynamic proteome, and demonstrate that this approach is applicable for tagging and (co-) localizing CDSs on a genome-wide scale.     

A novel method for determining linkage between DNA sequences: hybridization to paired probe arrays

Cooperative hybridization has been used to establish physical linkage between two loci on a DNA strand. Linkage was detected by hybridization to a new type of high-density oligonucleotide array. Each synthesis location on the array contains a mixture of two different probe sequences. Each of the two probes can hybridize independently to a different target sequence, but if the two target sequences are physically linked there is a cooperative increase in hybridization yield. The ability to create and control non-linear effects raises a host of possibilities for applications of oligonucleotide array hybridization. The method has been used to assign linkage in 50:50 mixtures of DNA containing single nucleotide polymorphisms (SNPs) separated by 17, 693, 1350 and 2038 bp and to reconstruct haplotypes. Other potential uses include increasing the specificity of hybridization in mutation detection and gene expression monitoring applications, determining SNP haplotypes, characterizing repetitive sequences, such as short tandem repeats, and aiding contig assembly in sequen-cing by hybridization.     

Chemical nanoprinting: a novel method for fabricating DNA microchips

We have developed a novel cost-effective procedure, namely ‘chemical nanoprinting’, for oligonucleotide or cDNA chips manufacture. In this thermo-controlled process, the oligonucleotides, covalently attached to a highly loaded ‘master-chip’ through disulfide bonds, are chemically transferred to the acrylamide layer mounted on a ‘print-chip’. It is demonstrated here that multiple identical print-chips can be produced from a single master-chip. This duplication process is a few hundreds of times faster than any existing methods and the speed of process and cost incurred are independent of the scale of the DNA chips.