Recently, I investigated the possibility of a novel fusion protein in lung tumors. Since I had sequence information on one fusion partner, I was able to verify the presence of the 3' end of its transcript with RT-PCR. I then wanted to know if the 5' end of the transcript was from the same protein or from a novel fusion partner. Since I had no
a priori knowledge of the sequence of the potential fusion partner, designing a primer pair to investigate this possibility was out of the question. In an attempt to determine the sequence upstream of my original RT-PCR amplicon, I chose to use a technique referred to as 5'-RACE, for Rapid Amplification of cDNA Ends.
My previous experience with the quality of the molecular biology enzymes and kits from Roche Applied Science led me to try their 5'/3'-RACE Kit, 2nd Generation (Catalog No. 03 353 621 001). The kit contains all the buffers, enzymes, dNTPs, and positive control components for 10 RACE experiments. Not supplied are Taq polymerase, a cDNA/PCR product purification kit, and gene-specific primers. The tubes of reagents in the kit are clearly numbered and color-coded to minimize mistakes.
Conceptually, 5'-RACE is straightforward but the procedure is complex enough to make the kit well worth ordering. Starting with total or poly(A)+ RNA, cDNA is generated using a gene-specific reverse primer (SP1) and Roche’s Transcriptor Reverse Transcriptase. After purifying the resulting cDNA, a poly(A) tail is added to the 3' end with the kit-supplied Terminal Transferase and dATP. The dA-tailed cDNA is then amplified using a second gene-specific primer (SP2), which hybridizes to a site 3' to SP1, and an oligo-dT anchor primer from the kit. In addition to the poly-dT region, the oligo-dT anchor primer contains a stretch of nucleotides that adds a multiple cloning site to the PCR product. If this PCR reaction generates sufficient product for sequence analysis, you’re done. If not, Roche recommends an additional round of PCR using their anchor primer and a third nested gene-specific primer (SP3), 3' to SP2.
In my case, the PCR reaction with the SP2/oligo-dT anchor primer pair generated plenty of product, which appeared as a smear between 0.6 and 1.3 kb. Since all members of this collection of amplicons were generated using SP2 and the primer hybridizing to the dA-tailed end of the cDNA, sequencing starting from the end opposite to the dA-tail was possible even though the sequencing reaction would contain DNA products of varying lengths. Although the results indicated that I did not have a novel fusion protein, the important point was that the kit allowed me to answer the question and move on to the next question.
An important feature of the 5'/3'-RACE Kit is the inclusion of a positive control RNA and pertinent primers. These can be included in the experimental RACE reactions for useful feedback as to the success of each step.