Labeled DNA

Fig. 3.46. " Sn image of Sn-labeled DNA hybridized to six oligonucleotide spots three complimentary and three non-comple-mentary.

A diagnostic method using fluorescence labeled DNA probes to detect and quantify the number complementary chromosomal sequences on a cellular resolution. A related technique that also allows assessment of gene amplifications, but without precise quantification of copy numbers is the chromogenic in situ hybridization (CISH). Here, instead of a fluorescent dye an enzyme that can generate a colored precipitate in the tissue samples is coupled to the DNA probe. 

The stoichiometry of the recharged DNA/PLL/SPLL particles was studied using sucrose-gradient ultracentrifugation of fluorescently labeled polyion complexes in 25 mM HEPES buffer. Rhodamine-labeled DNA (Rh-DNA) and either fluorescein-labeled PLL (Fl-PLL) or SPLL (Fl-SPLL) were used to determine their relative amounts within DNA... 

Nick translation A technique for labeling DNA based on the ability of the DNA polymerase from E colt to degrade a strand of DNA that has been nicked and then to resynthesize the strand if a radioactive nucleoside triphosphate is employed, the rebuilt strand becomes labeled and can be used as a radioactive probe. 

Southwestern hlot A method for detecting pro-tein-DNA interactions by applying a labeled DNA probe to a transfer membrane that contains a rena-tured protein. 

FISH. Fluorescent in-situ hybridization a method utilizing fluorescently labeled DNA probes to detect or confirm gene or chromosome abnormalities that are generally beyond the resolution of routine cytogenetics. 

A non-radioactively labeled DNA probe of 742bp was used for the hybridization. The probe was obtained from a genomic DNA fi om FORL r2 using the same oligonucleotide above mentioned and the same conditions. The... 

A site at the Agricultural Experimental Station (Ithaca, NY) was treated in microcosms with C-labeled glucose, phenol, caffeine, and naphthalene. Levels of C02 were measured to assess utilization of the substrates, and the populations analyzed by separating the C-labeled DNA by density centrifugation, followed by PCR amplification and sequencing of 16S rRNA (Padmanabhan et al. 2003). Populations contained relatives to a range of bacteria that varied with the substrate. Only relatives of Acinetobacter were found in all samples, and for caffeine only Pantoea. 

It has been established that methane is produced on rice roots by reduction of CO2. This was examined in rice roots using a combination of 16S rRNA sequencing and density gradient fractionation of C-labeled DNA after incubation with C02. The major groups of archaea detected were Methanosarcinaceae that decreased with time to be replaced by the hitherto uncultured Rice Cluster I, although the former subsequently dominated (Lu et al. 2005). 

While metal-nitrogen and metal-oxygen bonded compounds dominate nucleobase coordination chemistry, examples in which metal-carbon bonds are formed have been identified. Early studies on the synthesis of metal-labeled DNA demonstrated that nucleotide-triphosphates, UTP, CTP, dUTP, and dCTP, can undergo mercury modification at C5 (82,83). The UTP derivative was also shown to act as a substrate for RNA polymerase in the presence of mercaptans (83). Later, guano-sine was shown to undergo mercury modification at C8 though, in this case, the purine was multiply substituted, 21 (84). 

Maxam, A. M. Gilbert, W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980, 65, 499-560. 

A variation on this method, called fluorescent in situ hybridization (FISH), uses fluorescent-labeled DNA and RNA probes for detection and visualization of single cells by microscopy or flow cytometry.7 80 The FISH technique is popular because of its sensitivity and speed of visualization fluorescent dyes can be used to produce probes with different colors for simultaneous detection of several organisms.76,81,82... 

Following previous work, Hagiwara and collaborators [71] recently prepared 5 -terminal acridone-labeled DNAs, using the succinimidyl ester 24 of the acridone acetic acid 23 reported before [69], and evaluated their use as donors for a fluorescence resonance energy transfer (FRET) system in combination with 3 -dabcyl-tagged DNA... 

Shoji A, Hasegawa T, Kuwahara M et al (2007) Chemico-enzymatic synthesis of a new fluorescent-labeled DNA by PCR with a thymidine nucleotide analogue bearing an acridone derivative. Bioorg Med Chem Lett 17 776-779... 

DNA polymerase I has been purified to homogeneity. When the pure enzyme is treated with subtilisin, a proteolytic enzyme from Bacillus subtilis, the polymerase is cleaved into two pieces. The small fragment retains the 5 to 3 nuclease activity, whereas the larger piece, called a Klenow fragment, has both polymerase activity and the 3 to 5 exonuclease activity. The Klenow fragment is sold commercially for use in labeling DNA for use in detecting recombinant DNA. 

MapMarker sets of fluorescently labeled DNA fragments for sizing standards and compatible with (fluorescent-based) separation instruments systems. 

Deckert V., Zeisel D., Zenobi R., Vo-Dinh T., Near-field surface enhanced Raman imaging of dye-labeled DNA with 100-nm resolution, Anal. Chem. 1998 70 2646-2650. 

Probe the solution of labeled DNA that is hybridized with the array. 

TRITC has been used in numerous applications involving fluorescence detection, including double-staining techniques with fluorescein-labeled probes (Mossberg and Ericsson, 1990), the synthesis of fluorescently labeled DNA probes (Smith et al., 1985), as a label in homogeneous... 

The two-step nature of SPDP crosslinking provides control over the conjugation process. Complexes of defined composition can be constructed by adjusting the ratio of enzyme to secondary molecule in the reaction as well as the amount of SPDP used in the initial activation. The use of SPDP in conjugation applications is extensively cited in the literature, perhaps making it one of the more popular crosslinkers available. It is commonly used to form immunoto-xins, antibody-enzyme conjugates, and enzyme-labeled DNA probes. A standard activation and coupling procedure can be found in Chapter 5, Section 1.1. 

Re-dissolve the labeled DNA pellet in water and store at —20°C until used. 

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