Citrus sinensis flesh cDNA-AFLP Library

Vector: PGEM Teasy vector; Site_1: BstYI; Site_2: MseI; Fruits were harvested at three developmental stages. Poly-A mRNA was isolated from total RNA extracted from flesh according to the Trizol LS and Dynabeads M-280 Streptavidin protocols. A cDNA-AFLP technique of RNA fingerprinting was optimized allowing the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent polymerase chain reaction (PCR) amplification. This is based on the selective amplification of cDNA fragments obtained by digestion with one frequent and one rare cutter enzymes, followed by ligation of two double-stranded adapters of known sequences. AFLP-TP (Transcriptional Profiling) is an improved version of the cDNA-AFLP protocol, that allows the isolation one unique restriction fragment for each cDNA. The esacutter enzyme is BstYI (recognition site Pu/GATCPy) and the fourcutter is MseI (recognition site T/TAA). The difference in AFLP-TP lies in an additional step between the two digestions: as the cDNA synthesis is obtained with a biotinylated oligo-dT, this allows to collect the 3 end of the transcript after the first digestion, taking advantage of the high affinity of the biotin for the streptavidin anchored to magnetic beads (Dynal). After the electrophoretic run, a vertical scanner collected the fluorescence emissions derived from the excitation of the fluorescein molecules attached to the BstYI primers with a 465nm monochromatic beam. Single fragments were isolated either through DGGE (Denaturing Gradient Gel Electrophoresis) analysis or cloning into pGEM-T Easy vector (Promega). Samples that showed only one neat band were sequenced.