Technique / Molecular Biology / PCR / 5' and 3' RACE

Rapid amplification of cDNA end on 5' or 3' end.

BACKGROUND: The use of specific but partially degenerate primers for nucleic acid hybridisations and PCRs amplification of known or unknown gene families was first reported well over a decade ago and the technique has been used widely since then. RESULTS: Here we report a novel and successful selection strategy for the design of hybrid partially degenerate primers for use with RT-PCR and RACE-PCR for the identification of unknown gene families. The technique (named PaBaLiS) has proven very effective as it allowed us to identify and clone a large group of mRNAs encoding neurotoxin-like polypeptide pools from the venom of Agelena orientalis species of spider. Our approach differs radically from the generally accepted CODEHOP principle first reported in 1998. Most importantly, our method has proven very efficient by performing better than an independently generated high throughput EST cloning programme. Our method yielded nearly 130 non-identical sequences from Agelena orientalis, whilst the EST cloning technique yielded only 48 non-identical sequences from 2100 clones obtained from the same Agelena material. In addition to the primer design approach reported here, which is almost universally applicable to any PCR cloning application, our results also indicate that venom of Agelena orientalis spider contains a much larger family of related toxin-like sequences than previously thought. CONCLUSION: With upwards of 100,000 species of spider thought to exist, and a propensity for producing diverse peptide pools, many more peptides of pharmacological importance await discovery. We envisage that some of these peptides and their recombinant derivatives will provide a new range of tools for neuroscience research and could also facilitate the development of a new generation of analgesic drugs and insecticides.

3' Rapid amplification of cDNA ends (3' RACE) is a polymerase chain reaction (PCR) based technique which has been developed to analyse 3' ends of partially known cDNA sequences. To improve the effectiveness of the technique, many investigators have modified the RACE protocol. Here, we describe an alternative procedure for analysing 3' mRNA ends which is based on DNA ligase-mediated self circularization and inverse PCR. This technique is simple and characterized by the exclusive use of gene-specific primers and the absence of unspecific adaptor sequences to obtain highly specific PCR products. We applied the method to analyze the 3' UTR of human mono-ADP-ribosyltransferase (ART) 3 mRNA in testis and heart muscle and of ART4 mRNA in HEL cells. The obtained sequences of ART3 and ART4 mRNA corresponded to data base entries of the respective mRNAs. No adenylate/uridylate-rich elements (AREs) were found in the 3' UTR of ART3 mRNA while one ARE class I motif was detected in the 3' UTR of ART4 mRNA.

The sequencing of aromatase cDNA from rabbit granulosa cells was obtained by RACE PCR. This cDNA is 2.9 kb long. The first 119 nucleotides correspond to the first untranslated exon. Nucleotides 120 to 1,629 correspond to the coding region (1,509 nucleotides) and the rest of the sequence is non coding and contains a polyadenylation signal. Translation of the cDNA sequence indicates that the protein is composed of 503 amino acids, like in human aromatase. Its molecular weight is 57.4 kDa. The alignment between the rabbit aromatase amino acid sequence and other aromatases already described in the human, mouse, rat, cow, pig, chicken, rainbow trout and teleost fish shows that the rabbit protein exhibits the highest homology with the human one (85%).