Introduction

Rapid amplification of cDNA ends (RACE) is an efficient approach for obtaining full-length cDNA when only partial sequences are available (1,2,3). The principle of RACE is that an anchor sequence (to be used as the PCR primer binding template) is added to the end of the cDNA, followed by a series of hemi-nested anchored PCRs to generate the flanking sequences. Normally 5′ and 3′ RACE are performed separately. Because only one gene-specific primer is used, traditional 5′ and 3′ RACE often generate a high level of nonspecific amplification especially for the 5′ RACE. Several strategies, such as the CapSelect, step-out PCR, and solid phase cDNA synthesis, have been developed to eliminate background problems (2,3,4,5,6). These strategies depend on the so-called CapFinder approach (first described by Clontech on its web site), which is based on the ability of the Moloney murine leukemia virus (MMLV) reverse transcriptase to add 2–4 cytosine residues to the 3′ end of newly synthesized cDNAs upon reaching the 5′ end (cap region) of the messenger RNA (mRNA). When an oligonucleotide with 3–4 G residues (named the T-S oligo) at its 3′ end is present in the reverse transcriptase reaction, the T-S oligo can base pair with the 2–4 C residues of the newly synthesized cDNA. Reverse transcriptase then switches templates (from the mRNA to the oligonucleotide) and continues transcribing the oligonucleotide, thus attaching the complementary oligonucleotide sequence to the 3′ terminus of the cDNA ((Figure 1)). The advantage of the template-switching procedure is its simplicity, in that it avoids using a set of enzymatic reactions after the completion of first strand cDNA synthesis involved in most RACE approaches, such as homopolymeric tailing and ligation anchored tailing. However, during the reverse transcriptase reaction, the T-S oligo is free to anneal not only to the oligonucleotide stretch at the 3′ end of the cDNA, but also potentially to anywhere on the RNA acting as a primer for reverse transcription, which could cause heavy background if the T-S PCR primer is used in a subsequent PCR. PCR suppression (4) and solid phase cDNA synthesis protocols (5) could not totally eliminate the problems arising from contamination of the primers used for cDNA synthesis or/and nonspecific cDNAs generated by the CapFinder primer, because similar to other RACE methods, these approaches used only one gene-specific primer. Thus, PCR with two gene-specific primers should be a better way to solve the problem. Inverse PCR has been used in the amplification of flanking unknown sequences by using two primers pointing away from the known sequences (7,8,9).

Figure 1.

Here we report a modified reverse transcription inverse PCR approach for performing 5′ and 3′ RACE simultaneously. This approach combines the advantages of the template-switching effect (simplicity in obtaining full-length cDNAs and attaching an oligonucleotide sequence to the 3′ terminus of the full-length cDNAs) and inverse PCR (generation of a specific target product).

Materials and Methods

The organisms, genes, and oligonu-cleotides used in this study are listed in (Table 1). Total RNA was isolated using TRI Reagent® (Molecular Research Center, Cincinnati, OH, USA). All oligonucleotides were synthesized from Invitrogen (Carlsbad, CA, USA). First strand cDNA was synthesized with 1 µg total RNA. The reaction conditions were as follows: 10 pmol anchor oligo(dT) was annealed to 1 µg total RNA in a volume of 10 µL RNase-free water, by heating the mixture at 70°C for 10 min, followed by cooling on ice for 5 min. Transcription was then initiated by mixing the annealed RNA with 200 U PowerScript™ Reverse Transcriptase (Clontech, Mountain View, CA, USA) in a final volume of 20 µL containing 1× first-strand buffer, 10 mM dithiothreitol (DTT), 10 pmol T-S primer, and 1 mM each of dATP, dGTP, dCTP, and dTTP. The reaction was incubated at 42°C for 90 min. Aliquots (1–2 µL) of this reaction were subjected to a Platnum® Pfx DNA polymerase PCR system (Invitrogen) in 50 µL with the PCR anchor primer and T-S PCR primer ((Table 1)). The double-stranded cDNA products were purified with GlassMax® spin cartridges (Invitrogen). For phosporylation, 0.1 µg purified double-stranded cDNA was diluted to a concentration of 2 ng/µL in 1× ligation buffer, and 5 U T4 polynucleotide kinase (Fermentas, Hanover, MD, USA) were added. The reaction was incubated for 30 min at 37°C. The kinase was inactivated at 70°C for 10 min followed by a 2-fold dilution of the reaction mixture with 1× T4 ligase buffer to replenish the ATP. The intramolecular circularization was initiated by the addition of T4 DNA ligase (Fermentas) to a concentration of 4 U/100 µL, and the reaction was incubated at 4°C for 16 h.