Highly Regioselective C-5 Iodination of Pyrimidine Nucleotides and Subsequent Chemoselective Sonogashira Coupling with Propargylamine

An efficient C-5 iodination of pyrimidine-5′-triphosphates and subsequent palladium-catalyzed Sonogashira coupling reaction with propargylamine is described. The iodination reaction is highly regioselective and the coupling reaction is highly chemoselective that furnishes exclusive 5-(3-aminopropargyl)-pyrimidine-5′-triphosphate in good yield with high purity (>99%).     

Detection of groundnut rosette umbravirus infections with radioactive and non-radioactive probes to its satellite RNA

A cloned cDNA copy of the satellite RNA of groundnut rosette virus (GRV), labelled with either ³²P or digoxigenin, was used as a probe to detect the satellite RNA in infected leaves. The test was successfully applied to N. benthamiana and to groundnuts, infected with isolates of GRV from East and West Africa and with isolates which cause different types of symptom in groundnuts, including one which is almost symptomless. Although the probe did not react with extracts from plants infected with GRV isolates from which the satellite RNA had been artificially eliminated, all naturally occurring GRV isolates contain the satellite RNA. The test therefore provides a reliable indicator of infection by GRV.     

Amplified Fragment Length Polymorphisms (AFLPs) detected with non-radioactive digoxigenine labelled primers in three plant species

A protocol for the detection of AFLPs with the non radioactive digoxigenine labelling is presented. The protocol has been tested on DNA samples from three different plant species. The AFLP technique was used for the first time in these species. The sensitivity and reliability of the digoxigenine labelled primers in the AFLP technique was of the same order as the sensitivity and reliability of the radioactive assay. No major adjustments of the current standard AFLP protocols is necessary to use the digoxigenine labelled primers.     

基于荧光染料Cy5.5标记的引物延伸法鉴定细菌RNA剪切加工位点

【背景】传统引物延伸法常与放射性标记结合使用,但由于放射性元素半衰期短、危害人体健康及严格的操作要求等限制了其在一般实验室的广泛使用。因此,开发新型非放射性引物延伸法成为当前研究的热点。【目的】建立非放射性的引物延伸法,并利用该方法实现对细菌RNA剪切加工位点的鉴定。【方法】将包含RNA剪切位点的序列克隆至双荧光报告系统,然后利用荧光染料Cy5.5标记的特异性靶向mcherry寡核苷酸引物进行延伸,并辅以Northern blotting、RT-qPCR等技术,确定RNA剪切加工的精确位点。【结果】鉴定了来自解纤维梭菌cip-cel mRNA中的2个剪切加工位点均位于颈环结构下游的单链区域,且在剪切位点附近未发现保守序列。【结论】基于Cy5.5标记的引物延伸法能够精确鉴定RNA剪切加工位点,且与传统的放射性引物延伸法相比,该方法具有更高的安全性和更快的检测速度,能够满足一般实验室的实验要求。     

A protocol for non-radioactive DNA labelling and detection in the RFLP analysis of rice and tomato using single-copy probes

An improved protocol for non-radioactive labelling and detection of rice and tomato DNA is described. The procedure includes the use of polymerase chain reaction for the incorporation of digoxigenin-dUTP in the DNA molecule and the use of a chemiluminescent compound (AMPPD) for the signal detection.     

A Non-radioactive DNA Sequencing Method Using Biotinylated Dideoxynucleoside Triphosphates and {Delta}Tth DNA Polymerase

We synthesized a set of four biotinylated dideoxynucleosidetriphosphates (biotin-9-ddNTPs) and optimized the reaction conditionsfor non-radioactive cycle sequencing using modified Tth DNApolymerase (Tth) and a chemiluminescent detection system. Theresulting sequencing ladders showed lower background comparedto those with the conventional non-radioactive sequencing methodwhich uses 5'-biotinylated primers, especially when PCR productswere analysed. With our method, DNA sequences can be determinedat any primer positions without preparing 5'-biotinylated primersfor dideoxy chaintermination.     

Clinical performance of non-radioactive assays for HIV-1 DNA amplified by the polymerase chain reaction

A major factor preventing more widespread use of polymerase chain reaction in the clinical laboratory is the lack of convenient non-radioactive probe hybridization procedures which do not sacrifice sensitivity or specificity. In this report, we describe comparisons of probes labelled with biotin, digoxygenin, alkaline phosphatase, and³²P. We report the comparison of solution or liquid hybridization assay and Southern blotting with digoxygenin-labelled oligonucleotides on a total of 64 clinical specimens. Perfect diagnostic agreement between the³²P and digoxygenin probes was obtained. These data suggest that the non-radioactive assay as described is as sensitive and as specific as the assay with³²P-Iabelled probes.     

Expression and localization of the CYP2B subfamily predominantly in neurones of rat brain

Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone-metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone-sensitive areas and neurones (e.g. hippocampus) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.