Expression profiling by iAFLP: A PCR-based method for genome-wide gene expression profiling

The availability of comprehensive sets of genes has prompted the researcher s to carry out systematic collection of gene expression data. RT-PCR has th e highest specificity and sensitivity for transcript detection among all av ailable methods. Low throughput, especially when quantitative data are desi red, has precluded RT-PCR from genome-wide application. Here we report a PC R-based expression profiling method, introduced amplified fragment length p olymorphism (iAFLP), that has the same specificity and sensitivity as RT-PC R and a throughput level comparable to that of DNA-microarray hybridization . In this method, restricted ends of total cDNAs from six sources were liga ted with adaptors having various length of short insertions to a common seq uence (polymorphic adaptors). Amplification of a pool of these differential ly adapted cDNAs with a gene-specific primer and an adaptor primer allows u s to quantitate the abundance of any transcript in six mRNA sources. Using three different primer colors this technique allows quantitation of express ion of 864 genes across six different sources per day with a single autoseq uencer, which is comparable to the throughput of microarray analysis in ter ms of number of genes x number of sources.