A cDNA-AFLP protocol with reciprocally arranged 2-enzyme sequential digestion and silver staining detection

摘要

This study provides a modified protocol to the standard cDNA-amplified fragment length polymorphism (AFLP) procedure that has been used to detect differentially expressed transcripts in various systems. Modifications aimed to improve the overall coverage of the technique and the isolation of single fragments from each of the cDNA species. The protocol utilizes oligo-d(T) coupled magnetic beads to isolate and synthesize, and 2-enzyme sequential digestions of ds-cDNA molecules with flip-flop strategy on the beads to release transcript tags representing individual mRNA type and quantity. The protocol has been applied to detect Puccinia sorghi-induced expressional changes in maize leaf material sampled at 8 time points following inoculation. cDNA-AFLP analysis of this material pooled into 2 time intervals (6-24 h and 36-96 h), along with their controls, revealed banding patterns in which 10-20 differentially expressed message tags were present among the 40-80 bands detected per primer combination. Three hundred and ten differentially expressed message tags were sequenced, and the majority with known functions were found to be associated with plant-microbe interactions. Although it requires a few additional steps, this protocol appears to be effective in revealing differentially expressed messages, and the study demonstrates that pooling the material in the preparation of templates and silver staining detection provides economical alternatives for surveying and identifying expressional modulations occurring over relatively longer time periods.