Nucleic Acids Research

You, Yong; Moreira, Bernardo G.; Behlke, Mark A.; Owczarzy, Richard

doi: 10.1093/nar/gkl175pmid: 16670427

Locked nucleic acids (LNA) show remarkable affinity and specificity against native DNA targets. Effects of LNA modifications on mismatch discrimination were studied as a function of sequence context and identity of the mismatch using ultraviolet (UV) melting experiments. A triplet of LNA residues centered on the mismatch was generally found to have the largest discriminatory power. An exception was observed for G–T mismatches, where discrimination decreased when the guanine nucleotide at the mismatch site or even the flanking nucleotides were modified. Fluorescence experiments using 2-aminopurine suggest that LNA modifications enhance base stacking of perfectly matched base pairs and decrease stabilizing stacking interactions of mismatched base pairs. LNAs do not change the amount of counterions (Na+) that are released when duplexes denature. New guidelines are suggested for design of LNA probes, which significantly improve mismatch discrimination in comparison with unmodified DNA probes.

Schatz, Philipp; Distler, Jürgen; Berlin, Kurt; Schuster, Matthias

doi: 10.1093/nar/gkl218pmid: 16670426

The methylation of CpG dinucleotides has become a topic of great interest in cancer research, and the methylation of promoter regions of several tumor suppressor genes has been identified as a marker of tumorigenesis. Evaluation of DNA methylation markers in tumor tissue requires hundreds of samples, which must be analyzed quantitatively due to the heterogeneous composition of biological material. Therefore novel, fast and inexpensive methods for high throughput analysis are needed. Here we introduce a new assay based on peptide nucleic acid (PNA)-library hybridization and subsequent MALDI-TOF analysis. This method is multiplexable, allows the use of standard 384 well automated pipetting, and is more specific and flexible than established methods, such as microarrays and MS-SNuPE. The approach was used to evaluate three candidate colon cancer methylation markers previously identified in a microarray study. The methylation of the genes Ade-nomatous polyposis coli (APC), glycogen synthase kinase-β-3 (GSK3β) and eyes absent 4 (EYA4) was analyzed in 12 colon cancer and 12 normal tissues. APC and EYA4 were confirmed as being differentially methylated in colon cancer patients whereas GSK3β did not show differential methylation.

Adilakshmi, Tadepalli; Lease, Richard A.; Woodson, Sarah A.

doi: 10.1093/nar/gkl291pmid: 16682443

We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotron X-ray beam can footprint the tertiary structure and protein contacts of RNA–protein complexes with nucleotide resolution in living cells.

Seringhaus, Michael; Kumar, Anuj; Hartigan, John; Snyder, Michael; Gerstein, Mark

doi: 10.1093/nar/gkl184pmid: 16648358

Transposons are widely employed as tools for gene disruption. Ideally, they should display unbiased insertion behavior, and incorporate readily into any genomic DNA to which they are exposed. However, many transposons preferentially insert at specific nucleotide sequences. It is unclear to what extent such bias affects their usefulness as mutagenesis tools. Here, we examine insertion site specificity and global insertion behavior of two mini-transposons previously used for large-scale gene disruption in Saccharomyces cerevisiae: Tn3 and Tn7. Using an expanded set of insertion data, we confirm that Tn3 displays marked preference for the AT-rich 5 bp consensus site TA[A/T]TA, whereas Tn7 displays negligible target site preference. On a genome level, both transposons display marked non-uniform insertion behavior: certain sites are targeted far more often than expected, and both distributions depart drastically from Poisson. Thus, to compare their insertion behavior on a genome level, we developed a windowed Kolmogorov–Smirnov (K–S) test to analyze transposon insertion distributions in sequence windows of various sizes. We find that when scored in large windows (>300 bp), both Tn3 and Tn7 distributions appear uniform, whereas in smaller windows, Tn7 appears uniform while Tn3 does not. Thus, both transposons are effective tools for gene disruption, but Tn7 does so with less duplication and a more uniform distribution, better approximating the behavior of the ideal transposon.

Yang, Inchul; Park, In Young; Jang, Sung-Moon; Shi, Lian Hua; Ku, Hyung-Keun; Park, Sang-Ryoul

doi: 10.1093/nar/gkl257pmid: 16679448

We report a novel method for rapid quantification of the degree of DNA methylation of a specific gene. Our method combined bisulfite-mediated PCR and quantification of deoxyribonucleoside monophosphate (dNMP) contents in the PCR product through capillary electrophoresis. A specific bisulfite-PCR product was enzymatically hydrolyzed to dNMP monomers which were quantitatively analyzed through subsequent capillary electrophoresis. PCR following bisulfite treatment converts unmethylated cytosines to thymines while leaving methyl-cytosines unchanged. Then the ratio of cytosine to thymine determined by capillary electrophoresis represents the ratio of methyl-cytosine to cytosine in genomic locus of interest. Pure oligonucleotides with known sequences were processed in parallel as standards for normalization of dNMP peaks in capillary electrophoresis. Sources of quantification uncertainty such as carryovers of dNTPs or primers and incomplete hydrolysis were examined and ruled out. When the method was applied to samples with known methylation levels (by bisulfite-mediated sequencing) as a validation, deviations were within ±5%. After bisulfite-PCR, the analytical procedure can be completed within 1.5 h.

Guo, Yong-Chao; Zhou, Ya-Feng; Zhang, Xian-En; Zhang, Zhi-Ping; Qiao, Yan-Mei; Bi, Li-Jun; Wen, Ji-Kai; Liang, Mi-Fang; Zhang, Ji-Bin

doi: 10.1093/nar/gkl260pmid: 16682441

Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10 000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications.

Suzuki, Hitoshi; Zuo, Yuhong; Wang, Jinhua; Zhang, Michael Q.; Malhotra, Arun; Mayeda, Akila

doi: 10.1093/nar/gkl151pmid: 16682442

Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3′ to 5′ exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT–PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing.

Hamilton, G.; Brown, N.; Oseroff, V.; Huey, B.; Segraves, R.; Sudar, D.; Kumler, J.; Albertson, D.; Pinkel, D.

doi: 10.1093/nar/gkl160pmid: 16670425

We describe a charge-coupled device (CCD) imaging system for microarrays capable of acquiring quantitative, high dynamic range images of very large fields. Illumination is supplied by an arc lamp, and filters are used to define excitation and emission bands. The system is linear down to fluorochrome densities ≪1 molecule/µm2. The ratios of the illumination intensity distributions for all excitation wavelengths have a maximum deviation ∼±4% over the object field, so that images can be analyzed without computational corrections for the illumination pattern unless higher accuracy is desired. Custom designed detection optics produce achromatic images of the spectral region from ∼ 450 to ∼750 nm. Acquisition of a series of images of multiple fluorochromes from multiple arrays occurs under computer control. The version of the system described in detail provides images of 20 mm square areas using a 27 mm square, 2K × 2K pixel, cooled CCD chip with a well depth of ∼105 electrons, and provides ratio measurements accurate to a few percent over a dynamic range in intensity >1000. Resolution referred to the sample is 10 µm, sufficient for obtaining quantitative multicolor images from >30 000 array elements in an 18 mm × 18 mm square.

Kawasaki, H.; Suyama, E.; Iyo, M.; Taira, K.

doi: 10.1093/nar/gkl306pmid: 16670428

Hiroki, Tomoko; Song, Young-Han; Liebhaber, Stephen A.; Cooke, Nancy E.

doi: 10.1093/nar/gkl174pmid: 16648359

The human vitamin D-binding protein (hDBP) gene is a member of a cluster that includes albumin, α-fetoprotein and α-albumin genes. The common origin, physical linkage and hepatic expression of these four genes predict shared regulatory element(s). However, separation of hDBP from the other three genes by 1.5 Mb argues that hDBP may be under autonomous control. To test for hDBP autonomy, mouse lines were generated with a transgene containing the hDBP gene along with extensive flanking sequences. Expression of this transgene was hepatic, robust and proportional to transgene copy number. DNase I hypersensitive site (HS) mapping revealed five liver-specific HS at the hDBP locus: HSI and HSIII at −2.1 kb and −0.13 kb upstream of the transcription initiation site, HSIV and HSV within intron 1 and HSVII located 3′ to the poly(A) site. A second transgene with minimal flanking sequences confirmed the sufficiency of these gene-proximal determinants for hepatic activation. The hepatic-specific HS aligned with segments of phylogenetically conserved non-coding sequences. These data demonstrate the autonomy of the hDBP locus and suggest that this control is mediated by chromatin-based locus control determinants in close proximity to, and within the transcription unit.