CDNA probes

The induction of HSPs correlates with the increase of the levels of their transcripts. Liquid hybridisation studies conducted by Schoffl Key (1982) using cloned cDNA probes have revealed that about 20 different species of HSP18 mRNA accumulate to 19 000 copies per cell within two hours of heat... 

Drought also has a profound effect on protein synthesis. In many plant tissues, a reduced water potential causes a reduction of total protein synthesis and a rapid dissociation of polyribosomes. The latter has been shown not to be the consequence of increase in ribonuclease activity (Hsiao, 1973 Dhindsa Bewley, 1976). For a specific protein, Jacobsen, Hanson Chandler (1986) have shown in barley leaves that water stress enhances the synthesis of one of the a-amylase isozymes. Using a cDNA probe they found that water-stressed leaves contained much more a-amylase mRNA than unstressed plants. 

Grasp one end of a dust-free 22 mm X 40 mm microscope glass coverslip with forceps. Lower one end near the cDNA probe until it touches the surface outside the printed area and slowly lower the opposite end of the coverslip onto the slide. The solution will spread across the entire print area beneath the coverslip. Use a yellow tip to carefully adjust the position of the coverslip over the printed area. Large air bubbles can be moved away from the hybridization area by a gentle tapping on the coverslip with a yellow tip. Small air bubbles will be released during hybridization. 

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. 

Therefore, chemiluminescent methods using an acridinium ester-labeled cDNA probe allow the discrimination of a mismatched DNA sequence in a homogeneous assay. 

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

In the method shown in Figure 9A, a biotin-labeled cDNA probe is first immobilized to a polyvinylchloride microtiter plate well that is coated with bio-tinylated-bovine serum albumin [33], The target DNA is hybridized in the liquid-phase with a digoxigenin-labeled probe, so that the biotin-labeled probe can capture a marker enzyme. An antibody-conjugated enzyme is then added, followed by a chemiluminescent substrate. 

The acridinium ester (AE) in an AE-labeled cDNA probe hybridized to target DNA is less likely to be hydrolyzed than in the unhybridized conformation (Fig. 10) [9-11]. Single-base mismatches in the duplex adjacent to the site of AE attachment disrupt this protection, resulting in rapid AE hydrolysis [11]. Hydrolysis by a weak base renders AE permanently nonchemiluminescent. After hydrolysis, it is possible to use the remaining chemiluminescence as a direct measure of the amount of hybrid present. This selective degradation process is a highly specific chemical hydrolysis reaction, which is sensitive to the local environment of the acridinium ester. The matched duplex can be detected and quantified readily, whereas the mismatched duplex produces a minimal signal. 

Figure 10 Mismatch detection by using a chemiluminescent AE-labeled cDNA probe. Procedure [9, 11] Acridinium ester-labeled probes specific for either wild-type or mutant sequence corresponding to a target DNA are hybridized with the sample DNA for 1.0 h at 60°C in a hybridization buffer (pH 5.2). Hybridized and nonhybridized probes are discriminated by the hydrolysis reaction for 12 min at 62.5°C in the presence of Na2B407 (pH 8.5) and Triton X-100. The chemiluminescence of each sample is then measured in a luminometer.

This protocol permits detection of 10 pmol of target DNA dotted on a nylon membrane after hybridization with the d(G)30 probe [15]. The background chemiluminescence caused by nonspecific binding of the probe in the hybridization buffer to the membrane is negligible in this assay system. However, both the target DNA and the cDNA probe bound to the membrane are detected solely... 

Figure 11 Chemiluminescent detection for membrane hybridization of unmodified DNA target by derivatization reaction with TMPG. Procedure [15] A portion of the DNA solution is spotted on a nylon membrane. The target DNA is hybridized to its cDNA probe having a -(G)15TT(G)15TT at its 3 terminus in a hybridization buffer (pH 7.0) at 42°C for 2 h. After washing, the membrane is moistened with sodium phosphate solution (pH 10) for a few seconds, and then immersed in 0.2 M TMPG dissolved with dimethyl sulfoxide for 0.5 min at ambient temperature. The moist membrane is then dipped in dimethyl-formamide for a few seconds, and the luminescence is detected for 0.5 min.

The advantages of detecting cDNA probes by chemiluminescence and bioluminescence include high sensitivity and simple protocols, using either manual film... 

Incubation periods in excess of 2 h were required before this activity was detected in cell-free supernatants. More recently, the use of cDNA probing of Northern transfers (to detect specific mRNA levels), the use of ELISA techniques (to detect protein levels immunologically) and the development of more specific bioassays (culture techniques in which a biomolecule stimulates proliferation in a particular cell line) have resulted in a more thorough analysis of IL-1 production by neutrophils. IL-1 is only poorly expressed in blood neutrophils because mRNA for this cytokine is detectable only at very low levels (if at all), and protein production is usually below the level of detection of most assays. However, exposure of neutrophils to lipopolysaccharide (LPS), or to cytokines such as GM-CSF, TNF or IL-1 itself, results in a rapid but transient increase in IL-1 expression. 

If a single-stranded DNA molecule is placed with a complementary single DNA sequence the two molecules will hybridize. This hybridization forms the basis of a number of very powerful techniques for detecting and quantifying specific nucleic acid sequences. The hybridization may be carried out either in solution or more commonly with the DNA immobilized on nitrocellulose filters. The complementary DNA sequence is known as a cDNA probe. Probes for a large number of important nucleic acid sequences are now available. 

The specificity of the hybridization between probe and DNA depends on the temperature and ionic strength of the buffer. At high temperatures and low salt concentration (high stringency), hybridization is very specific. At lower stringencies (low temperatures and high salt concentration) the hybridization is less specific and the cDNA probe will bind to many sequences. In Southern... 

Another similar technique to Southern blotting is Northern blotting. Here, instead of DNA fragments, mRNA fragments are probed with a labelled cDNA probe after separation by electrophoresis and transfer to nitrocellulose membranes. Northern blotting is used to detect and quantify mRNA from tissue extracts. 

Figure 13.15 Detection of a specific sequence of DNA by hybridization to a 32P-labelled cDNA probe. DNA is transferred to nitrocellulose and incubated with the probe. After washing, specific binding is visualized by autoradiography. The DNA sequence detected by the probe is present in lanes 2, 3 and 5 but not 1 and 4.

The enzyme DNA polymerase 1 is used in the production of labelled cDNA probes... 

Pieces of DNA, for example genes or cDNA probes, are multiplied using a method based on in vivo replication in bacterial cells. The sequence of DNA is incorporated into a vehicle or vector which transports it into a cell host, usually E. coli. As the bacteria culture grows, the vector also replicates so producing more of the required sequence of DNA. Two of the naturally occurring types of DNA molecule which can be used as vectors are plasmids and viral chromosomes. 

A combination of pulse field gel electrophoresis to separate large DNA restriction fragments, amplification by PCR, and detection with p53 cDNA probes has been useful to study chromosomal deletions and p53 mutations in colorectal cancers (Bl). 

---