http://hdl.handle.net/2144/2436 Sat, 25 May 2013 14:33:16 GMT 2013-05-25T14:33:16Z http://hdl.handle.net/2144/3372 End Invasion of Peptide Nucleic Acids (PNAs) with Mixed-Base Composition into Linear DNA Duplexes Smolina, Irina V.; Demidov, Vadim V.; Soldatenkov, Viatcheslav A.; Chasovskikh, Sergey G.; Frank-Kamenetskii, Maxim D. Peptide nucleic acid (PNA) is a synthetic DNA mimic with valuable properties and a rapidly growing scope of applications. With the exception of recently introduced pseudocomplementary PNAs, binding of common PNA oligomers to target sites located inside linear double-stranded DNAs (dsDNAs) is essentially restricted to homopurine–homopyrimidine sequence motifs, which significantly hampers some of the PNA applications. Here, we suggest an approach to bypass this limitation of common PNAs. We demonstrate that PNA with mixed composition of ordinary nucleobases is capable of sequence-specific targeting of complementary dsDNA sites if they are located at the very termini of DNA duplex. We then show that such targeting makes it possible to perform capturing of designated dsDNA fragments via the DNA-bound biotinylated PNA as well as to signal the presence of a specific dsDNA sequence, in the case a PNA beacon is employed. We also examine the PNA–DNA conjugate and prove that it can initiate the primer-extension reaction starting from the duplex DNA termini when a DNA polymerase with the strand-displacement ability is used. We thus conclude that recognition of duplex DNA by mixed-base PNAs via the end invasion has a promising potential for site-specific and sequence-unrestricted DNA manipulation and detection. Tue, 04 Oct 2005 00:00:00 GMT http://hdl.handle.net/2144/3372 2005-10-04T00:00:00Z http://hdl.handle.net/2144/3196 Simultaneous Quantitative and Allele-Specific Expression Analysis with Real Competitive PCR Ding, Chunming; Maier, Esther; Roscher, Adelbert A; Braun, Andreas; Cantor, Charles R BACKGROUND. For a diploid organism such as human, the two alleles of a particular gene can be expressed at different levels due to X chromosome inactivation, gene imprinting, different local promoter activity, or mRNA stability. Recently, imbalanced allelic expression was found to be common in human and can follow Mendelian inheritance. Here we present a method that employs real competitive PCR for allele-specific expression analysis. RESULTS. A transcribed mutation such as a single nucleotide polymorphism (SNP) is used as the marker for allele-specific expression analysis. A synthetic mutation created in the competitor is close to a natural mutation site in the cDNA sequence. PCR is used to amplify the two cDNA sequences from the two alleles and the competitor. A base extension reaction with a mixture of ddNTPs/dNTP is used to generate three oligonucleotides for the two cDNAs and the competitor. The three products are identified and their ratios are calculated based on their peak areas in the MALDI-TOF mass spectrum. Several examples are given to illustrate how allele-specific gene expression can be applied in different biological studies. CONCLUSIONS. This technique can quantify the absolute expression level of each individual allele of a gene with high precision and throughput. Wed, 05 May 2004 00:00:00 GMT http://hdl.handle.net/2144/3196 2004-05-05T00:00:00Z http://hdl.handle.net/2144/3034 Tuning and Controlling Gene Expression Noise in Synthetic Gene Networks Murphy, Kevin F.; Adams, Rhys M.; Wang, Xiao; Balázsi, Gábor; Collins, James J. Synthetic gene networks can be used to control gene expression and cellular phenotypes in a variety of applications. In many instances, however, such networks can behave unreliably due to gene expression noise. Accordingly, there is a need to develop systematic means to tune gene expression noise, so that it can be suppressed in some cases and harnessed in others, e.g. in cellular differentiation to create population-wide heterogeneity. Here, we present a method for controlling noise in synthetic eukaryotic gene expression systems, utilizing reduction of noise levels by TATA box mutations and noise propagation in transcriptional cascades. Specifically, we introduce TATA box mutations into promoters driving TetR expression and show that these mutations can be used to effectively tune the noise of a target gene while decoupling it from the mean, with negligible effects on the dynamic range and basal expression. We apply mathematical and computational modeling to explain the experimentally observed effects of TATA box mutations. This work, which highlights some important aspects of noise propagation in gene regulatory cascades, has practical implications for implementing gene expression control in synthetic gene networks. Mon, 08 Mar 2010 00:00:00 GMT http://hdl.handle.net/2144/3034 2010-03-08T00:00:00Z http://hdl.handle.net/2144/3028 Labeling of Unique Sequences in Double-Stranded DNA at Sites of Vicinal Nicks Generated by Nicking Endonucleases Kuhn, Heiko; Frank-Kamenetskii, Maxim D. We describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3′ terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3′-flap DNA is obtained. The single-stranded terminus in 3′-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic λ-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA. Tue, 01 Apr 2008 00:00:00 GMT http://hdl.handle.net/2144/3028 2008-04-01T00:00:00Z