Nonradioactive probes detection

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... 

Nucleic acids can also be biotinylated by nonenzymatic methods with photobiotin, a photoactivatable biotin analog (6), which can be commercially obtained from BRL, Sigma, and other commercial sources I have not compared the suitability of this method of biotin incorporation with that reported here, but expect that the method would be fully acceptable FMC (Rockland, ME) markets an alternate nonradioactive sequence detection kit known as Chemiprobe. The basis of this system is a chemical modification of cytosine residues m the probe DNA. After hybridization, the probe is detected by means of a monoclonal antibody that specifically recognizes the sulfonated DNA. Detection of the bound monoclonal antibody is achieved by means of an alkaline phosphatase-conjugated second antibody. 

Presently, nonradioactive probes, especially biotin or digoxigenin, are favored because they are less hazardous to work with, can be more rapidly developed, and provide better spatial resolution. Thus, introduction of nonradioactive detection systems has made ISH, using formalin-fixed and paraffin-embedded tissues, more accessible for application to molecular cell biology and diagnostic pathology. However, radioactive detection systems are more sensitive than nonradioactive probes, especially oligonucleotide probes used instead of cRNA probes (Sperry et al., 1996). 

The controversy over the degree to which radioactive probes are more sensitive has not been fully resolved. In any case, microwave pretreatment enhances ISH signal detection of RNA and DNA whether radiolabeled or nonradioactive probes are used both methods are presented later. Furthermore, a number of approaches is available to increase the sensitivity of the nonradioactive ISH procedures (for a review, see Komminoth and Werner, 1997) some of these approaches are discussed below. 

Compared with radioactive ISH, nonradioactive ISH requires a 10- to 50-fold higher concentration of probes such as oligonucleotides. However, signal amplification is decreased by increasing probe concentration. Therefore, since nonradioactive probes have limited sensitivity, especially when applied to low-abundance mRNAs, a technique is required for signal amplification. One such technique consists of an optimized protocol for rapid signal amplification based on catalyzed reporter deposition (CARD) that increases the sensitivity of nonradioactive mRNA ISH on the formaldehyde-fixed and paraffin-embedded tissues (Speel et al., 1998). This technique facilitates the detection of low-copy mRNAs by ISH (Yang et al., 1999). 

An acridinium ester-labeling method has been developed by Gen-Probe, Inc.. Because of high quantum yield and flash reaction kinetics in the presence of base and H2O2, chemiluminescent acridinium esters provide the possibility of designing sensitive nonradioactive probes. The detectability of these systems is 5 x 10 mol and acridinium-labeled probes are fully compatible with hybridization. Arnold et al. (1989) discriminated hybridized from unhybridized acridinium ester-labeled DNA probes without prior separation (Fig. 7.3). In a typical experiment (Table 7.7), the ss probe is hybridized to... 

Hartley et al. (1986) devised a nonradioactive probe, called probe-vector, which can, after hybridization, transform competent E. coli cells and, depending on the transformation efficiency, detect as little as 0.1 pg of target nucleic acid. The probe-vector molecules are linear, partially si DNA prepared by hybridizing individually prepared DNA strands. The ds region of the probe-vector encodes a phenotypic marker and origin of replication. The two terminal si... 

Use 0.5 ml/cm and a stringency which yields optimum signal/noise ratios while maintaining the desired detectability. The stringency can be adjusted by the ionic strength, the temperature of the wash and the concentration of SDS. For oligonucleotides and nonradioactive probes, 0.5% Tween 20 can be used instead of SDS. The hybridization solution is first collected (can often be reused), followed for radioprobes by a hot rinse , to remove the bulk of radioactivity, at room temperature. 

Nonradioactive probes require adaptations of detection to solid phase format. For instance, nonspecific binding of antibody or enzyme may differ greatly between nitrocellulose and nylon or the conditions may have to be adjusted to permit the reaction product to precipitate in situ (e.g. for TMB). 

Despite the wide use of radioprobes in colony or plaque hybridization assays, nonradioactive probes can be advantageous. The use of biotinylated probes, initially the most common among nonradioactive detection systems, is limited since biotin-streptavidin systems tend to give high background levels with bacterial material unless specific measures are taken. The more recently developed DIG (Table 7.2), but also other hapten-antibody systems such as sulfonated probes, are very attractive alternatives. The main restriction is that monoclonal antibodies (commercially available) should be used since polyclonal antisera often contain antibodies against bacteria. The main drawback of nonradioactive probes is the ability to reprobe the same membrane. It is possible, however, to strip a membrane of its probe after a colorimetric detection and to perform a chemiluminescent detection or vice versa. 

Chapters 17-22 describe the hybridization of the nonradioactive probes to the DNA and RNA immobilized on blots, together with the detection systems necessary to reveal where the probe has hybridized. Chapters 17-19 deal with digoxigenin probes, with Chapters 17 and 19 describing chemiluminescent detection on DNA and RNA blots respectively, and Chapter 18 describing a colorimetric detection system. Chapter 20 deals with enhanced chemiluminescent detection of enzymically labeled probes, whereas Chapters 21 and 22 describe enhanced chemiluminescent detection of large (Chapter 21) and small (oligonucleotide. Chapter 22) probes labeled with fluorescein. 

Chapters 28 and 29 describe the use of nonradioactive probes to detect gene transcripts in thin sections in situ. Chapter 30 describes the use of nonradioactive probes on whole embryo mounts. The probes used in this section are digoxigenin-labeled RNA probes, and the detection is colorimetric, revealing the cell and tissue types that are expressing particular genes. 

Slides with nonradioactive probes are now ready for hybrid detection (see Protocol 7.5). Slides with radioactive probes should be dehydrated through ethanol, as in step Ic, and air-dried for autoradiography (see Protocol 7.6). 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

Ruth, J.L. (1993) Direct attachment of enzymes to DNA probes. In Methods in Nonradioactive Detection (G.C. Howard, ed.), pp. 153-177. Appleton and Lange, Norwalk, Connecticut. 

In situ hybridization techniques are used to subtype the papilloma virus that may be found in premalignant lesions in uterine cervix. Most of the techniques use nonradioactively labeled avidin-biotin probes. A number of specific biotin-labeled probe cocktails are available for HPV subtype identification (e.g., HPV 6-l l, 16-18, 31-33-35). Some of these techniques use chemiluminescent components to enhance the sensitivity of HPV subtype detection (H5). 

Three types of detection methods can be used for in situ hybridization radioactive, fluorescent, and chromogenic. Radioactive detection techniques are the most commonly used primarily because they are more sensitive, but also because the other techniques are relatively more recent in terms of the development of the appropriate chemistry and the techniques that enable in vitro labeling of probes by these nonradioactive means. 

In the tissue sections, a hybridization is performed with a highly specific probe in order to detect the target (either amplified by indirect in situ RT-PCR or unamplified). For the preparation of nonradioactive digoxigenin-labeled probes, one should refer to the guidelines of the company manuals Genius M System User s guide for membrane hybridization and Nonradioactive in situ hybridization application manual (2nd ed., Boehringer Mannheim). 

Detection of the sites of hybridization-dependent binding of biotinylated probe to the filters is most readily conducted with commercially available kits. Favorable results have been obtained with the BluGene Nonradioactive Nucleic Acid Detection System from BRL. Follow the manufacturer s instructions when carrying out the following steps. After washing, sequentially expose the filters to streptavidin and biotinylated alkaline phosphatase (or to a conjugate of these two proteins) This causes the immobilization of alkaline phosphatase at sites of positive hybridization... 

Related introns with considerable sequence similarity may be detected by heterologous hybridization with labeled probes. Restriction fragments of suitable length to cover almost the entire sequence of a cloned intron are labeled radioactively or by alternative, nonradioactive methods. We use the following protocol for random primed DNA labeling, derived from Feinberg and Vogelstein.14... 

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