Labeling DNA probes

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. 

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... 

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. 

Fluorescently labeled DNA probes can be used for detection, localization, or quantification of target DNA sequences. In situ hybridization mapping of genomic DNA sequences can be... 

The second label also may be a fluorescent compound, but doesn t necessarily have to be. As long as the second label can absorb the emission of the first label and modulate its signal, binding events can be observed. Thus, the two labeled DNA probes interact with each other to produce fluorescence modulation only after both have bound target DNA and are in enough proximity to initiate energy transfer. Common labels utilized in such assay techniques include the chemiluminescent probe, N-(4-aminobutyl)-N-ethylisoluminol, and reactive fluorescent derivatives of fluorescein, rhodamine, and the cyanine dyes (Chapter 9). For a review of these techniques, see Morrison (1992). 

Habili, N., Mclnnes, J.K., and Symons, R.H. (1987) Non-radioactive photobiotin-labelled DNA probes for the routine diagnosis of barley yellow dwarf virus. J. Virol. Meth. 16, 225-237. 

Leary, J.J., Waldrop, A.A., and Ward, D.C. (1983) Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose Bio-blots. Proc. Natl. Acad. Sci. USA 80, 4045 1049. 

These blotting techniques are known by the names of compass directions (Southern, Northern, Western). Since Southern is a person s name, there s no logic in how the different blots were named. Southern developed a blot in which DNA on the blot is detected by a labeled DNA probe. It was then fairly logical that the next technique developed, detecting RNA on the blot with a DNA probe, should be called a Northern blot. Then things got carried away with the Western, and now the Southwestern, and so on and so on. 

Acridinium esters have also been utilized for chemiluminescent detection of cDNA probes (Fig. 5) [9-11], The hydrolysis rate is much faster when the ester is conjugated to single-stranded DNA, rather than to double-stranded DNA. This means that the chemiluminescence from unhybridized acridinium ester-labeled probe is rapidly lost, whereas the chemiluminescence from the hybridized probe is minimally affected. This permits discrimination between hybridized and unhybridized acridinium ester-labeled DNA probes without separation steps. 

Advances in the fluorescence in situ hybridization (FISH) technique with the ability to label DNA probes with as many as six different spectrally distinct... 

A3. Arnold, L. J., Jr., Hammond, P. W., Wiese, W. A., and Nelson, N. C., Assay formats involving acridinium-ester-labeled DNA probes. Clin. Chem. (Winston-Salem, N.C.) 35, 1588-1594(1989). 

Hapten-labeled DNA probes for FISH assays can be used for BISH assays and those probes can be purchased from various vendors. If commercial hapten-labeled DNA probes are not available, DNA probes can be labeled with haptens by nick translation or by PCR in a laboratory. Nick translation kit can be utilized for labeling DNAprobes with hapten (10976776001, Roche Applied Science, Germany). New probes must be analyzed for the specificity by FISH or BISH assays using a comparative genomic hybridization metaphase control slide. 

Common haptens used for labeling DNA probes for BISH assays are biotin, DIG, DNP, FITC, and Texas Red. Based on the size of your DNA targets, you may choose from a direct detection or an indirect detection for BISH assays. In general, an indirect detection system can provide better sensitivity compared to a direct detection system. For an indirect detection, you need to select a combination of two antibodies raised with two different animal species, such as a mouse anti-DIG antibody and a rabbit anti-DNP antibody, so that enzyme-labeled anti-mouse antibody and anti-rabbit antibody can be applied for signal detection. If a direct BISH detection is going to be applied, anti-hapten antibodies raised in the same animal species that are labeled with either AP or HRP enzyme molecules... 

ZytoVision ZytoDot2C GISH Implementation Kit contains all necessary BISH detection reagents for the detection of DIG-and DNP-labeled DNA probes on formalin-fixed tissue sections. The probes can be purchased from ZytoVision or other vendors. 

Fig. 28. Synthesis of labeled DNA probes. A Labeled DNA can be generated using different enzymes (Klenow fragment of DNA polymerase or a terminal transferase) to incorporate labeled nucleotides into specific DNA sequences. Probes can be labeled using radioactive nucleotides or nucleotides labeled with an immunogenic molecule such as biotin. B The labeled probe is then hybridized to the target nucleic acid, which is either bound to a membrane or in a tissue section or cell. An antibody is then used to detect the non-radioactively-labeled probe. C The antibody may be conjugated to a fluorescent or chemiluminescent dye, or an enzyme that produces a color reaction. The target nucleic acid is thus visualized.

Large numbers of clones obtained can be screened rapidly by colony hybridization using a labeled DNA probe. Thus, if it is desired to isolate a gene for a particular protein and some part of that protein has... 

In addition to biotin, a digoxigenylated derivative of dUTP was also synthesized. This derivative of dUTP can be incorporated into DNA by Pol I (or the Klenow fragment of Pol I). Therefore, digoxigenin-labeled DNA probes can be prepared by nick translation or random primed-labeling methods developed for the biotin system. It is almost certain that more nonradioactive alternatives to biotin and digoxigenin will be developed in the future. Chemiluminescent methods for nonradioactive probe detection are now widely being used... 

DNA is denatured and transferred from the agarose gel to a cellulose nitrate sheet. The DNA firmly bound to the sheet is hybridized with a radioactively labeled DNA probe, which carries some of the sequences of interest. The radiolabel, which hybridizes to specific regions of the sheet, is detected by autoradiography. By comparing the results obtained from the DNA of different individuals one can see if the labeled DNAs move with the same or a different mobility. If they move differently, there must be a RFLP difference between the individuals. Detection of an RFLP by this means usually depends on the restriction enzyme used in the initial digestion. Some enzymes show a difference others do not. 

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