An efficient and site-specific gene trimming method

Trimming a DNA strand into a precisely determined fragment can be carried out efficiently by an improved method involving a site-specific trim-primer and a single-stranded DNA template which is generated from a multifunctional vector, pTZ18R, and linearized by using an Eco RI-pTZ18R splinter. A complementary DNA strand is synthesized by DNA polymerase I (Klenow), and the 3'-end of the template upstream from the annealed primer is trimmed by subsequent T4 DNA polymerase reaction. An ATG translation initiator codon or a termination codon can be incorporated into the trim-primer, providing versatility to this single-stranded DNA-initiated gene trimming method that can be applied to subcloning and expression of any DNA fragment with known terminal sequences.     

An efficient method for matching nucleic acid sequences.

A method of computing the fraction of matches between two nucleic acid sequences at all possible alignments is described. It makes use of the Fast Fourier Transform. It should be particularly efficient for very long sequences, achieving its result in a number of operations proportional to n ln n, where n is the length of the longer of the two sequences. Though the objective achieved is of limited interest, this method will complement algorithms for efficiently finding the longest matching parts of two sequences, and is faster than existing algorithms for finding matches allowing deletions and insertions. A variety of economies can be achieved by this Fast Fourier Transform technique in matching multiple sequences, looking for complementarity rather than identity, and matching the same sequences both in forward and reversed orientations.     

Micronuclear DNA sequences of Oxytricha fallax homologous to the macronuclear inverted terminal repeat.

The macronucleus of the protozoan Oxytricha fallax is generated from a micronucleus following conjugation. While the micronucleus contains high molecular weight DNA, the macronucleus contains only short linear DNA molecules which all end in the same 20 bp inverted terminal repeat (Ma-ITR). The Ma-ITR was radioactively labeled and purified for use as a probe in hybridizations to micronuclear and macronuclear DNA. Sequences homologous to the Ma-ITR were detected in micronuclear DNA. The copy number of the repeat in the micronuclear genome is approximately that required to encode the macronuclear DNA termini. The micronuclear copies are found embedded in repeated long sequence blocks.     

NMR and CD studies on an oligonucleotide containing N4-methylcytosine.

The hexamer d(CGm4CGCG) exists predominantly as a right handed B form helix at 20 degrees C in 150 mM NaCl, as shown by 2D NOE spectra. Under these conditions a minor species is also observed which corresponds to the single strand in slow exchange on a proton NMR time scale with the double strand. This exchange is unusually slow and separate resonances for the two species are seen up to 65 degrees C. At 50 degrees C the lifetime of the single strand species is 0.85 s. Under high salt conditions the hexamer is partly converted into the Z form, but the complete transition is only observed at 5M NaCl at -6 degrees C.     

An efficient method for finding repeats in molecular sequences.

The problem of finding repeats in molecular sequences is approached as a sorting problem. It leads to a method which is linear in space complexity and NlogN in expected time complexity. The implementation is straightforward and can therefore be used to handle large sequences with relative ease. Of particular interest is that several sequences can be treated as a single sequence. This leads to an efficient method for finding dyads and for finding common features of many sequences, such as favorable alignments.     

An efficient technique for obtaining sequences flanking inserted retroviruses.

Genomic mapping studies frequently employ retrovirus-mediated transfer of dominant selectable markers to specific target chromosomes. DNA probes containing sequences adjacent to inserted proviruses are valuable mapping tools in such studies. We have implemented a strategy for amplification of chromosomal sequences flanking the 5' LTR of MoMuLV-based vectors. Probes derived from these amplification products successfully differentiated murine versus human proviral localization in retrovirus-infected mouse-human chromosome 17q hybrid cells.     

An efficient and versatile approach for the construction of oligonucleotide microarrays

A new immobilization chemistry for covalent attachment of phosphorylated oligonucleotides on epoxy-activated glass surface via opening of oxirane ring is described. The proposed strategy results in excellent immobilization efficiency, spot homogeneity, and morphology. The constructed microarray was successfully demonstrated for discrimination of nucleotide mismatches.     

An SMC-like protein is required for efficient homologous recombination in Arabidopsis.

In plants, the observed low frequency of gene targeting and intrachromosomal recombination contrasts markedly with the efficient extrachromosomal recombination of DNA. Thus, chromatin accessibility can have a major influence on the recombination frequency of chromosomal DNA in vivo. An Arabidopsis mutant hypersensitive to a range of DNA-damaging treatments (UV-C, X-rays, methyl methanesulfonate and mitomycin C) is also defective in somatic intrachromosomal homologous recombination. The wild-type gene encodes a protein closely related to the structural maintenance of chromosomes (SMC) family involved in structural changes in chromosomes. Although loss of SMC function is lethal in other eukaryotes, growth of the Arabidopsis mutant is normal in the absence of genotoxic treatments. This suggests a surprisingly specialized function for this protein in plants, and provides the first in vivo evidence for the involvement of an SMC protein in recombinational DNA repair. It is possible that SMC-like proteins in plants alleviate suppressive chromatin structure limiting homologous recombination in somatic cells.     

An efficient solid-phase strategy for the construction of chemokines.

Synthesis of chemokines via stepwise SPPS approaches has been shown to be a challenge. Herein, a complete study of different coupling methods, solvents and temperature combined with a continuous-flow synthesizer equipped with feedback monitoring was carried out. The results from this study indicate that this family of molecules can be prepared using an Fmoc/Bu(t) chemical approach and provide a general method to apply for the elongation of other difficult sequences.     

An efficient and economic enhancer mix for PCR

Polymerase chain reaction (PCR) has become a fundamental technique in molecular biology. Nonetheless, further improvements of the existing protocols are required to broaden the applicability of PCR for routine diagnostic purposes, to enhance the specificity and the yield of PCRs as well as to reduce the costs for high-throughput applications. One known problem typically reported in PCR experiments is the poor amplification of GC-rich DNA sequences. Here we designed and tested a novel effective and low-cost PCR enhancer, a concentration-dependent combination of betaine, dithiothreitol, and dimethyl sulfoxide that broadly enhanced the quantitative and/or qualitative output of PCRs. Additionally, we showed that the performances of this enhancer mix are comparable to those of commercially available PCR additives and highly effective with different DNA polymerases. Thus, we propose the routine application of this PCR enhancer mix for low- and high-throughput experiments.     

An oligonucleotide microarray for detection and discrimination of orthopoxviruses based on oligonucleotide sequences of two viral genes

An oligonucleotide microarray for detection and identification of orthopoxviruses was developed. Genus specific and orthopoxvirus species-specific regions of the genes encoding chemokine binding and alpha/beta-interferon binding proteins were used as a target. The developed microarray allows the variola, monkeypox, cowpox, vaccinia, camel-pox and ectromelia (mousepox) viruses to be distinguished with a high degree of reliability.     

Preparation and characterization of a uniformly 2 H/ 15 N-labeled RNA oligonucleotide for NMR studies.

An RNA oligonucleotide that contains the binding site for Escherichia coli ribosomal protein S8 was prepared with uniform 15N isotopic enrichment and uniform deuterium enrichment at all non-exchangeable sites using enzymatic methods. The RNA binding site, which contains 44 nt, forms a hairpin in solution and requires Mg2+for proper folding. The longitudinal magnetization recovery rates of the exchangeable protons were compared for the [2H,15N]-enriched RNA molecule and for the corresponding fully [2H,15N]-enriched RNA hairpin. It was found that 1H-1H dipolar relaxation significantly contributes to the recovery of exchangeable proton longitudinal magnetization. The exchangeable proton resonance line widths were less affected by deuteration, indicating that chemical exchange with H2O remains the dominant mechanism of transverse magnetization relaxation. Nevertheless, deuteration of this RNA hairpin was found to enhance the sensitivity of NOE-based experiments relative to the fully protonated hairpin and to simplify 2D NMR spectra. The increased signal-to-noise ratio facilitated the assignment of the cytidine amino resonances and several of the purine nucleotide amino resonances and permitted the identification of NOE crosspeaks that could not be observed in spectra of the fully protonated RNA hairpin.     

Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of alpha-synuclein

Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of alpha-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies. Based on NMR spectral data collected at a 920 MHz proton frequency, we performed epitope mapping of an anti-alpha-synuclein monoclonal antibody, and furthermore, characterized conformational effects of phosphorylation at Ser129 of alpha-synuclein.     

Super pairwise alignment (SPA): an efficient approach to global alignment for homologous sequences.

Sequence analysis is the basis of bioinformatics, while sequence alignment is a fundamental task for sequence analysis. The widely used alignment algorithm, Dynamic Programming, though generating optimal alignment, takes too much time due to its high computation complexity O(N(2)). In order to reduce computation complexity without sacrificing too much accuracy, we have developed a new approach to align two homologous sequences. The new approach presented here, adopting our novel algorithm which combines the methods of probabilistic and combinatorial analysis, reduces the computation complexity to as low as O(N). The computation speed by our program is at least 15 times faster than traditional pairwise alignment algorithms without a loss of much accuracy. We hence named the algorithm Super Pairwise Alignment (SPA). The pairwise alignment execution program based on SPA and the detailed results of the aligned sequences discussed in this article are available upon request.