Over the summer, I spent ample time preparing for the laboratory work necessary for this project. I researched the RACE protocol, which is the rapid amplification of cDNA ends. I began to understand how the protocol works and the necessary steps that must be taken. This process begins with RNA which is extracted from fresh or frozen tissue. Then, a specific oligo is added to the 5' end in place of the cap structure to allow easy identification of the region between the RNA cap and the start of the coding region. The RNA is reverse transcribed into cDNA using a reverse transcriptase. Then, the specific DNA sequences of interest can be amplified using a gene specific primer and a primer specific to the oligo added or an oligo dT primer for specific for 3' poly A tail. With these amplified regions, the DNA can be sequenced directly or cloned first and then sequenced. This will provide information on the 5' and 3' untranslated sequences that are upstream and downstream to the genes of interested. The three genes I am studying are from the Glycine max genome. The identifiers are glyma01g05050.1, glyma02g02430.1, and glyma18g16170.1. These genes have yet to be characterized and that is the bulk of what I have been working on to date. They are classified as WRKY genes, which have been studied in Arabidopsis thaliana and appear to be involved in defense mechanisms against disease and illness.
My work this semester began with RNA extraction from root tissue of an 18-day old w82 glycine max plant and root tips of a 3-day old w82 glycine max seedling. This extraction was performed with a Qiagen RNeasy Plant Mini Lit and followed by a DNA free treatment to purify the RNA obtained and remove any DNA or DNase contamination. Next, I utilized the Invitrogen GeneRacer Kit to perform the RACE protocol using only the RNA from the root tissue to start. This kit included all the necessary reagents to oligo cap the RNA and reverse transcribe it into cDNA. In addition, I designed 5' and 3' gene specific primers for each WRKY gene of interest based on the DNA sequence. With these gene specific primers and primers specific for the oligo and 3' poly A tail, I attempted to amplify the upstream and downstream regions of these genes. This was not as successful as originally hoped and much optimization was needed in the PCR reaction. I had to adjust the annealing temperature and dilute the DNA to a more manageable concentration in order to successfully amplify these regions. Ultimately, I was only able to obtain the 3' downstream regions of the glyma01g05050.1 and glyma18g16170.1 genes, which was verified by performing gel electrophoresis with a slow melt 1% agarose gel. The slow melt gel was used because it allowed extraction of the gel bands to be used in TOPO cloning. This is the step that I am currently at and will be working on next. By cloning these sequences into the TOPO vector, I will then be able to sequence them.
I was able to determine that the 5' capping was not successful with the use of two different positive control genes, Actin 1 and Beta Tubulin 1. Therefore, I must attempt again to perform this procedure with the GeneRacer kit. This will be performed within the next week or two. Hopefully, this second attempt is more successful and will allow the amplification and identification of the 5' upstream regions. As for the glyma02g02430.1 gene I will design new primers at different locations. I hope this will allow the PCR amplification to be more successful.
