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Combination of cross-species RNA solution hybridization and immunoprecipitation aids in the cloning of RT-PCR products.

cDNA cloning by means of reverse transcription-polymerase chain reaction (RT-PCR) is a widely used technique. Ever since the first published method by Veres et al. (6), it has supplanted conventional methods of cDNA cloning due to its ease and speed. However, there are certain constraints that are associated with this technique; e.g., it requires the knowledge of a few nucleic acid sequences of the gene of interest. These can be obtained by either microsequencing the protein itself followed by the construction of a putative cDNA sequence or, more commonly, by using the homologous cDNA sequence from other species. However, both methods use degenerate primers (4,5), which can give rise to nonspecific priming (2). Therefore, variations of PCR have been developed to overcome this problem, e.g., touchdown PCR (3) and nested PCR (1). However, none of the techniques has been used successfully in our laboratory to clone the rabbit serotonin (5-hydroxytryptamine, 5HT) type 3 receptor from the retina using degenerate primers based on a rat sequence (unpublished sequence data; GenBank Accession No. U59672). Although additional manipulations of PCR methods and reagents were attempted, none of these was successful in obtaining the rabbit 5-HT3 receptor gene coding regions. Indeed, very often, gel analysis of PCR products showed multiple bands (Figure 1A), none of which, after subsequent cloning and sequencing, was homologous to the 5-HT3 gene. Thus, our difficulties could, in part, arise because the level of 5-HT3 receptor RNA in the rabbit retina is extremely low. Furthermore, the degenerate primers could also have annealed with other more abundant genes that can serve as fortuitous templates. Consequently, even though the 5-HT3 transcripts could be amplified by this method, they might be obscured by amplification of the other more abundant genes that are amplified at the same time. This prompted us to devise an alternative that would improve the specificity of cDNA cloning using degenerate primers. Our method is based on the premise that cross-species pairing for long stretches of the same genes is sufficiently high; e.g., between either RNA and DNA or DNA and DNA strands. Once the duplex is formed, it can be selectively retrieved by immunoprecipitation. Thus, if a gene sequence is known in one species, appropriate RNA or DNA probes can be generated and used to identify and isolate the same gene in different species. Although this method is given in detail for our gene of interest, it can be easily adapted for a broad range of applications by those molecular biologists interested in the evolution of specific genes, comparative studies of gene structure and some clinical applications, e.g., the simultaneous identification of multiple strains of infectious agents such as human immunodeficiency virus (HIV). First, rat 5-HT3-digoxiginin (Dig)labeled RNA probes were generated using in vitro transcription with a DIG RNA Labeling Kit (SP6/T7) (Boehringer Mannheim, Indianapolis, IN, USA). A cloning vector with a rat 5HT3 insert that we had previously cloned (unpublished data) was used. Next, standard RT-PCR on rabbit retina RNA was performed using degenerate primers with the exception that the annealing temperature was slightly decreased to make sure that the gene of interest, in our case, the rabbit 5-HT3 receptor, was present in the PCR product. The RNA isolation was performed using RNAzol B (Tel-Test, Friendswood, TX, USA) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using SUPERSCRIPT II RNase HReverse Transcriptase (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s instructions. The PCR was: 2.5 μL of 20× Enzyme Buffer (1.0 M Tris-HCl, pH 9.0, 0.4 M ammonium sulfate), 1 μL dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, dTTP), 0.5 μg of each of the primers and 1 mM MgCl2. The reaction was run in a PTC-100 Thermal Cycler (MJ Research, Watertown, MA, USA) as follows: 94°C for 4 min (hot start), 35 cycles of 94°C for 45 s (denaturation), 50°C for 45 s (annealing) and