5'-RACEing across a bridging oligonucleotide.

Rapid amplification of cDNA 5′ ends (5′-RACE) is a convenient way to retrieve information about the 5′ termini of mRNAs because entire open reading frames are difficult to predict from genomic information and expressed sequence tag libraries generally lack full 5′ termini. To achieve this, numerous methods have been developed, each of which has been successful in specific situations (3–5,11–14,17). However, two major generic drawbacks are associated with these procedures. First, a general non-gene-specific primer is introduced for PCR amplification that can lead to nonspecific amplification products (3,5,11–14,17). Second, the methods used to introduce the nongene-specific primer sequence into the cDNA can be problematic. Some protocols involve ligation of ssDNA by T4 RNA ligase, the efficiency of this reaction being quite low (4,11). Others use terminal deoxynucleotidyl transferase (TdT) to tail the first-strand cDNA (5,11,12) and require additional steps, such as cDNA purification before and after tailing, which reduces the recovery of cDNA. Furthermore, the TdT tailing itself can be inefficient and difficult to control. To circumvent these drawbacks, we have devised a novel and effective method to amplify cDNA 5′ ends, termed BO-5′RACE, for 5′RACEing across a bridging oligonucleotide (BO). During an examination of the structural proteins residing within the peritrophic matrix, a structure lining the insect midgut of Mamestra configurata (Bertha armyworm), we isolated a 1.8-kb clone from a midgut cDNA library that bore a high degree of similarity to the Trichoplusia ni mucin (16). Northern blot studies revealed that the full-length transcript was approximately 3.5 kb. To obtain the complete mucin cDNA sequence, we developed the BO5′RACE protocol described below. Total RNA was isolated from midgut tissues of fourth instar larvae using TRIZOL® Reagent (Invitrogen, Carlsbad, CA, USA). The partial sequence of the mucin cDNA was used to design oligonucleotides (Table 1) for reverse transcription (RT), the BO, and PCR (F1, F2, R1, and R2) to test the BO-5′ RACE protocol (Figure 1). Notably, the RT primer is 5′-phosphorylated to allow for the head-to-tail ligation of the first-strand cDNA. A restriction endonuclease recognition site can also be introduced into the 5′ end of the RT primer if cloning of the cDNA 5′ terminus is required. The standard first strand cDNA synthesis reaction consisted of 5 μg total RNA, 2 pmol RT primer, first-strand buffer (50 mM TrisHCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 10 mM DTT, 6 mM MgCl2, 0.5 mM each dNTP, and 200 U SUPERSCRIPT II (Invitrogen) in a 20μL reaction. This mixture was incubated at 42°C for 60 min, and the reverse transcriptase was inactivated by incubation at 70°C for 15 min. The remaining mRNA was eliminated with the addition of 1 μL RNase H (Invitrogen) and incubation at 37°C for 1 h to preBenchmarks