A 18-mer partial phosphorothioate oligonucleotide sequence was synthesized and grafted in 5′ with a tyramine group which was
further radioiodinated. The Namalwa (VH 1 family) and HL-60 cell lines were transducted with liposome-mediated 125I-FR1-ASON. Liposome-mediated 131I-FR1-ASON and 131I labeled sense oligonucleotides were injected intratumorally into tumor-bearing BALB/c mice (6 weeks after inoculation of
107 Namalwa cells). Biodistribution was monitored by sequential scintigraphy and organ radioactivity measurement at 24 hours
after injection. The transduction efficiency in Namalwa cell lines reached (26.8±1.54)% that was higher than HL-60 cell lines.
Antisense probe images show tracer accumulation in tumor.
A vastagbéldaganatok korai diagnosztikája, terápiája, követése napjainkban sem teljesen megoldott. Munkánk során a vastagbéldaganatok biomarker-vizsgálatát, génexpressziós elemzését és osztályozását végeztük. Munkacsoportunk vizsgálatai alapján megállapítható, hogy a biopsziás minták oligonukleotid microarray-vizsgálati módszere az Affymetrix minőségi kritériumoknak messzemenően megfelelő, standardizált és jól reprodukálható. Megállapítottuk továbbá, hogy a Taqman mikrofolyadék-kártyarendszer alkalmas a chipen mért génexpressziós változások nagy teljesítményű, gyors és költséghatékony valós idejű PCR-es megerősítésére. Kimutattuk azt is, hogy az oszteopontin és az oszteonektin fokozatosan emelkedő mRNS- és fehérjeexpressziója erősen korrelál a vastagbél adenoma-dysplasia-carcinoma szekvencia előrehaladásával. Tíz, a colorectalis adenoma-dysplasia-carcinoma átmenettel párhuzamosan fokozódó mRNS-expressziójú, valós idejű PCR-rel is megerősített szöveti markert azonosítottunk, amelyet még nem írtak le az irodalomban. Vizsgálataink során 27, 13, illetve 10 „top” gént azonosítottunk, amelyek mRNS-expressziós mintázata a vastagbél-adenomával, a colorectalis carcinomával, illetve a gyulladásos bélbetegségekkel asszociálódik.
Authors:M. Jiang, Z.Q. Xaio, S.L. Fu, and Z.X. Tang
Fluorescence in situ hybridization (FISH) can reveal minor structural differences of chromosomes. The karyotype of common wheat (Triticum aestivum L.) based on FISH pattern is seldom reported. In this study, non-denaturing FISH (ND-FISH) using Oligo-pSc119.2-1, Oligo-pTa535-1 and (AAG)6 as probes was used to investigate the chromosomal structure of 85 common wheat including 83 wheat-rye 1RS.1BL translocation cultivars/lines, a wheatrye 1RS.1AL translocation cultivar Amigo and Chinese Spring (CS). Two, three, two, three, six, three and four structural types respectively for 1A, 2A, 3A, 4A, 5A, 6A and 7A chromosomes were observed. Two, eight, two, two, four and six types of chromosome for 2B, 3B, 4B, 5B, 6B and 7B were respectively detected. The structure of 1B chromosomes in Amigo and CS is different. Five, two, two and two types of chromosomal structure respectively for 1D, 2D, 3D and 5D were distinguished. Polymorphisms of 1RS.1BL, 4D, 6D and 7D chromosomes were not detected. Chromosomes 1AI, 2AI, 3AI, 4AI, 5AIII, 6AI, 7AIII, 2BI, 3BV, 4BI, 5BII, 6BIII, 7BI, 1DIV, 2DI, 3DI and 5DII appeared in these 85 wheat cultivars/lines at high frequency. Each of the 85 wheat cultivars/lines has a unique karyotype. Amigo is a complex translocation cultivar. The FISH karyotype of wheat chromosomes built in this study provide a reference for the future analyzing wheat genetic stocks and help to learn structural variations of wheat chromosomes. In addition, the results in this study indicate that oligonucleotide probes and ND-FISH technology can be used to identify individual wheat cultivar.
Authors:C. Giancola, A. Buono, G. Barone, L. De Napoli, D. Montesarchio, D. Palomba, and G. Piccialli
In this work we report a thermodynamic characterization of stability and melting behaviour of two 24-mer DNA triplexes. The
third strand, that binds the Watson-Crick double helix with Hoogsteen hydrogen bonds, contains 3′-3′ phosphodiester junction
that determines the polarity inversion. The target double helix is composed of adjacent and alternate fragments of oligopurine-oligopyrimidine
tracts. The two helices differ from the substitution of the cytosine, involved in the junction, with the thymine. Calorimetric
data reported here provide a quantitative measure of the influence of pH and base modification on the stability of a DNA triplex.
Authors:Saboki Ebrahim, T. Sharma, K. Usha, and Bhupinder Singh
Mango malformation is one of the most important diseases limiting its cultivation. However, disease resistance is known in some mango cultivars and is a desirable trait that can be utilized for developing mango varieties resistant to malformation. Resistance genes cloned from different plant species have revealed several similarities in DNA sequence and structural motifs. This provides the possibility of isolating resistance genes by polymerase chain reaction (PCR) with degenerate oligonucleotide primers designed from highly conserved regions of the nucleotide binding site (NBS). In the present study, we used eight combinations of oligonucleotide primers designed on the basis of P-loop and hydrophobic domains of conserved NBS-leucine rich repeat (LRR) protein sequences for amplifying resistance gene analogues (RGAs) in eight mango cultivars and hybrids showing a variable degree of resistance to mango malformation disease. A single band of about 500 bp in all mango cultivars was obtained from the s2+as2 primer combination. RGAs isolated from mango showed 73% similarity with RGAs in databases. It confirms that RGAs were actually isolated from mango. The obtained sequence can be used for isolating full length R-genes.
Authors:S. Omanwar, J. R. Rao, G. Butchaiah, S. H. Basagoudanavar, and R. K. Singh
The mechanically transmitted haemoflagellate,Trypanosoma evansicauses 'surra', a wasting disease of domestic animals and is highly endemic in distribution in Southeast Asia. The detection ofT. evansiis important for improving the epizootiological and animal health status of the region. The specificity and sensitivity of polymerase chain reaction (PCR) using oligonucleotide primers constructed fromT. evansirepetitive DNA sequences were studied in the present investigation. Using the assay, it was possible to amplify template DNA ofT. evansiderived from buffaloes, camels and horses to a threshold sensitivity level of 0.5 pg and to detect DNA from as few as five organisms in 10 (l crude blood samples. Following experimental infection of calves with 5 × 105T. evansi, positive signals could be observed as early as 12 h post-infection. DNAs from two common haemoflagellates of cattle,Babesia bigeminaandTheileria annulatawere not amplified with the primers.
Authors:L. Sámi, Krisztina Ursu, J. McKillen, S. Kecskeméti, S. Belák, and I. Kiss
Specific oligonucleotide primers were selected and combined in a multiplex arrangement, in order to detect simultaneously three economically important porcine viruses by polymerase chain reaction (PCR). The pathogen panel was comprised of viruses that cause reproductive failure in infected herds: Aujeszky’s disease virus (ADV), porcine parvovirus (PPV) and porcine respiratory and reproductive syndrome virus (PRRSV). In order to reduce the time required for the detection of the pathogens, the assay was optimised to a RapidCycler PCR instrument. The multiplex PCR assay was shown to be specific, sensitive and rapid, because the results were read in less than 60 min after sample preparation. Due to its speed, efficiency and sensitivity, the described rapid multiplex PCR assay serves as a useful novel tool in the veterinary diagnostic laboratories for the quick and complex detection of these important porcine pathogens.