Abstract: We have developed a 1-step procedure for cloning multiple bands obtained through cDNA–amplified fragment length polymorphism (AFLP) experiments. This protocol enables us to clone differential and nondifferential transcript-derived fragments (TDFs) in the same experiment. It consists of a mass cloning of cDNA-AFLP amplification products, identification of clones of interest with electrophoresis, and characterization through sequencing. The entire experiment can be performed in 1 wk because of the reduced time and effort needed for cloning, and it can be applied to any PCR method that produces complex band patterns. This method, tested on a comparison of gene expression between globular and heart embryo stages in sunflower embryogenesis, enabled us to clone 10 differential TDFs in the same experiment.
Key words: cDNA-AFLP, embryogenesis, sunflower, TDF
Abbreviations: AFLP, amplified fragment length polymorphism; HPLC, high-performance liquid chromatography; RT-PCR, reverse transcriptase-polymerase chain reaction; TDF, transcript-derived fragment; X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactoside.