Mass cloning of differential and nondifferential transcript-derived fragments from cDNA-AFLP experiments in sunflower

• Published in: Plant molecular biology reporter Vol. 22; no. 2; pp. 165 - 171
• Main Authors: Tamborindeguy,C, Ben, C, Jardinaud, F, Gentzbittel, L, Liboz, T
• Format: Journal Article
• Language: English
• Published: New York Springer Nature B.V 01-06-2004
• Subjects: Amplified fragment length polymorphism; Cloning; cloning (plants); complementary DNA; Embryogenesis; Embryonic growth stage; Experiments; Fragmentation; Fragments; Gene expression; Helianthus annuus; oilseed crops; Polymorphism; Transcription; transcription (genetics); transgenic plants
• ISSN: 0735-9640; 1572-9818
• DOI: 10.1007/BF02772723

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.