Nonradioactive probes

Biotin is a small vitamin molecule (Mr 244) that binds with highaffinity (kD 10 I5M I) to avidin. Avidin is a larger glycoprotein molecule (Mr 70,000), mostly distributed in egg whites. This protein has the advantage of conjugating with different markers, including peroxidase, fluorescent dyes, colloidal gold, and ferritin. Because of this property it is 

The avidin-biotin system was developed for detecting antigens at the electron microscope level (Heitzmann and Richards, 1974). Later Heggeness and Ash (1977) proposed the use of this system for fluorescence immunohistochemistry. Guesdon et al. (1979) proposed a variety of labeled avidin-biotin methods which were further supported by Warnke and Levy (1980). The avidin-biotin methods used today are similar to the system described by Hsu et al. (1981). This system is a significant improvement over the previous immuno-histochemical techniques. The problem of endogenous biotin is discussed on page 98. 

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). 

---

For nonradioactive probe flnorescence or enzymatic color development For radioactive probe dip in photographic emulsion, expose, develop, stain Microscopic examination... 

Because the disposal of radioactive waste is becoming increasingly expensive, nonradioactive probes have been developed. One of the most successful is based on the vitamin biotin (see p. 379), which can be chemically coupled to the nucleotides used to synthesize the probe. Biotin was chosen because it binds very tenaciously to... 

Isaac, P. G., ed. (1994) Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana Press, Totowa, New Jersey... 

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... 

With the advent of nonradioactive probes during the past decade, in situ hybridization has become an immensely valuable tool in the hands of ... 

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... 

A different approach to generate strand-specific probes is offered by lambda exonuclease (lambda exo Table 7.17C). It has 5 - 3 exonuclease activity but only from 5 -phosphoryl termini (5 -hydroxyl termini are resistant) (Little, 1981). One of the two strands can selectively bear a 5 -phosphoryl group by different approaches, such as phosphorylation of one of the two primers prior to PCR or successive restriction, dephosphorylation and restriction steps. In the first approach, the primers are removed after PCR cycling and the strand with the 5 -phosphoryl group digested from the duplex. The phosphorylated or its 5 -P-less equivalent can then be used to generate a strand-specific probe. Synthesis is achieved as described above. 7.6.5. Preparation of nonradioactive probes by PCR... 

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... 

As a general rule, prehybridization conditions (buffer and temperature) are, except for ohgonucleotide probes and charged nylon, identical to those of hybridization although the time is shorter (usually fast but prolonged prehybridization does not harm). Most (pre)hybridization buffers are variants of two basic buffers, one with formamide and one without. Formamide allows the use of lower incubation temperatures and an easy adjustment of the stringency and is generally used, or even essential, for RNA hybridization or for nonradioactive probes. It is recommended, particularly when formamide is used, to include a buffer, such as sodium/potassium phosphate, Tris-HCl, or PIPES-NaOH, or to use SSPE instead of... 

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). 

Hybridization of Northern blots calls for complicated buffers (Chapter 8). However, as for DNA (Church and Gilbert, 1984), it is possible to use single buyers with high SDS concentrations for (pre)hybridization (Virca et al., 1990). (Pre)hybridization is carried out in a solution containing 50 mM PIPES, 1 mM EDTA and 5% SDS for 15 min and hybridization in the same (fresh) buffer containing 10 cpm/ml of probe (or equivalent nonradioactive probe)... 

---