CONSTRUCTION AND CHARACTERIZATION OF A NORMALIZED CDNA LIBRARY

We have developed a simple procedure based on reassociation kinetics t hat can reduce effectively the high variation in abundance among the c lones of a cDNA library that represent individual mRNA species. For th is normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After c ontrolled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial dupl exes at relatively low Cot, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction a nd electroporated into bacteria, to propagate a normalized library wit hout a requirement for subcloning steps. An evaluation of the extent o f normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of oc currence of any clone examined in the normalized library was brought w ithin the narrow range of only 1 order of magnitude.