Nucleic acid labeling with

Polyplex uptake is typically assessed by transfecting cells with polyplexes formed with nucleic acids labeled with a fluorescent tag (such as rhodamine, fluorescein, and the alexa fluor dasses of dyes). " In some cases, the polymer or dendrimer can also be labeled with a fluorophore and cellular internalization of both the polymer and the nudeic acid is monitored. The assessment is then typically made by one or more of the following techniques measurement of the cell lysate fluorescence, fluorescence microscopy (wide-field or confocal miaoscopy)... 

Fig. 1 Cycloaddition reactions employed in nucleic acid labeling with reporter groups (green star). A Cu -mediated azide-alkyne cycloaddition (CuAAC) of a terminal alkyne with an azide. B Strain-promoted azide-alkyne cycloaddition (SPAAC) of an azide with a cyclooctyne derivative. C Staudinger ligation of an azide with a phosphine derivative (not a cycloaddition reaction, see below). D Norbornene cycloaddition of a nitrile oxide as 1,3-dipole and a norbornene as dipolarophile. E Inverse electron-demand Diels- Alder cycloaddition reaction between a strained double bond (norbornene) and a tetrazine derivative. F Photo-cUck reaction of a push-pull-substituted diaiyltetrazole with an activated double bond (maleimide)...

Chemical modification of nucleic acids with functionalized linkers, such as amine-functionalized alkyl chains tethered to nucleic acids, has been employed to modify surfaces with DNA probes [26]. Conductive supports and particularly Au-surfaces were functionaUzed with nucleic acid labeled with an oUgothymine thio-phosphate [(Ts ) ] [27] or with a thiol-functionalized linker tethered to the probe... 

Administration203 to chicks of acetic acid labeled with C14 (in the carboxyl group) gave D-glucose (from the glycogen) labeled equally at C3 and C4, but the D-ribose (from nucleic acids) had more label at C3 than at C2, indicating that in vivo D-ribose does not arise exclusively from hexose by loss of Cl. The 20-30% isotope content of D-ribose formed from D-glucose-l-C14 by Escherichia coli indicates that direct conversion is the major pathway but that a part is probably derived from transketolase action.204 206... 

In situ hybridisation Localizes specific genes within cells with nucleic acid-labelled probes Sensitivity and specificity dependent upon tissue and probe quality Yes... 

Fluorescein and its derivatives are one set of fluorescent labels that are commonly used to monitor DNA hybridization, and are often used as labels but do not selectively associate with the structure of double-stranded DNA. The most popular fluorescein derivative for nucleic acid labelling is FAM (car-boxyfluorescein). The structure of FAM is shown in Fig. 3. 

DNA labeled with ULYSIS Alexa Fluor 488, using a Nucleic Acid Labeling Kit (Invitrogen). DNA provided in the kit or oligonucleotides from other suppliers (e.g., Sigma) can be used. 

Another common enzyme used m these procedures is alkaline phosphatase. Alkaline phosphatase will cleave phosphates off of a donor molecule, which then in turn acts as a mediator of a color change involving a third molecule. This system is often used because alkaline phosphatase can create more of the color-producing molecules per enzyme molecule than can peroxidase, resulting in better sensitivity. Alkaline phosphatase systems are especially sensitive for examining protein or nucleic acid blots with enzyme labels. The problem when examining tissue is the presence of the endogenous enzyme m the tissues examined. 

Table 7.4 summarizes the characteristics of radionuclides most often used in nucleic acid labeling. Commercial dNTP preparations usually contain 10 (xCi/jxl and those with higher specific activities thus have a lower concentration. For instance, if the final radioactive concentration in the mixture is 1 jjcCi/p-I, the concentration of that radiolabeled dNTP is 1 piM if the specific activity is 1000 Ci/mmol but 0.2 p,M if the specific activity is 5000 Ci/mmol. 

Nucleic acid labelling, detection and quantitation with single fluorescent dye... 

The hybridization buffer, blocking agent, and detection reagents are available in an optimized kit form, including the reagents for labeling probes directly with HRP. (ECL direct nucleic acid labeling and detection system, RPN 3000, RPN 3001, RPN 3005, Amersham International, Amersham, UK). 

They are usually synthesised with reactive groups, R, on either one or both of the nitrogen atoms so that they can be chemically linked to nucleic acids or proteins. Used mainly for protein and nucleic acid labelling. Cy3 and Cy5 are commonly combined for 2 colour detection Cy3 dyes fluoresce yellow-green while Cy5 dyes fluoresce red. 

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

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