A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes

Abstract

Rapid amplification of cDNA ends (RACE) is widely utilized to determine the 5′-and 3′-terminal nucleotide sequences of genes. Many different RACE methods have been developed to meet various requirements, but none address the difficult problems that arise when trying to isolate the ends of extremely GC-rich genes. In this study, we found that we were unable to isolate the correct 5′ or 3′ ends of an insect gene, which appeared to include extremely GC-rich sequences, using current RACE methods. Thus, we developed a new RACE method that can be used for this purpose. This new method entails first strand cDNA synthesis at 70°C with Thermo-X™ reverse transcriptase in the presence of homoectoine, followed by a polymerase chain reaction with 98°C denaturation steps and Phusion™ DNA polymerase in the presence of 1M betaine and 5% DMSO. The use of these conditions yielded 5′-and 3′-RACE products that were about 80% GC over 213 and 162 bp, respectively, and included shorter, internal regions of 82-89% GC.

Alternative splicing of the sheep MITF gene: Novel transcripts detectable in skin

“…Also, it was difficult to PCR amplify the GC-rich 5′ end sequences. We solved this issue by either designing high Tm primers or following the use of homoectoine, a potent PCR enhancer (Schnoor et al, 2004) during the 5′ RACE cDNA synthesis protocol (Shi and Jarvis, 2006). Further, we identified four other MITF 5′ UTRs, commonly known as MITF-M ( Fig.…”

Characterization of a hydrophobin of the ascomycete Paecilomyces farinosus

“…If an oligo with an oligo(rG) overhang on its 3'end (a so called 'template switching oligo' -TSO) is present, it will hybridize with the poly C of the newly synthesized cDNA. Subsequently, the reverse transcriptase 'switches' to the newly added template and elongates it to the end [14]. For this approach custom made DNA oligonucleotide (CMO, Table 1) was ribo-tailed with rG using rGTP and terminal deoxynucleotidyl transferase (TdT), thus creating the TSO-primer (20 U TdT, 1 × TdT buffer, 0,4 mM rGTP, 25 pmol CMO, 40 U ribo-lock; 37 °C for 30 min, then 70 °C for 10 min).…”

“…Starting from the sequences obtained a classical 5′-RACE approach was performed according to [17]. A TSO ('template switching oligos' [14]) approach aiming at the 5′-end of the corresponding gene was also conducted. Only when using the latter protocol the complete coding sequence of pfah1 could be determined and the primary structure of the protein deduced.…”

Characterization of a novel mouse brain gene (mbu-1) identified by digital differential display

“…In many clones, however, application of the EST-based strategy to construct full-length cDNAs has not been successful (Carninci et al, 2003). In such cases, alternative strategies, such as directed cloning based on known gene sequences, are needed (Shi and Jarvis, 2006). These studies may eventually offer important clues for elucidating the cellular function of the corresponding proteins (Bono et al, 2003;Yu et al, 2003).…”