CHROMOSOME-SPECIFIC C-DNA LIBRARIES - REDUCTION OF UNSPECIFIC PRIMINGEVENTS BY PURIFICATION OF HETERONUCLEAR RNA

Chromosome specific c-DNA libraries greatly facilitate the isolation o f disease associated genes which have been previously linked to partic ular chromosomes. Recently, several methods have been developed and em ployed for the isolation of transcribed sequences from specific human chromosomes and chromosome regions. Heteronuclear (hn) RNA from somati c human/rodent cell hybrids has been used as starting material to sele ctively prime the synthesis of human specific c-DNAs. A drawback of th is method is the high number of rodent clones found in these chromosom e specific c-DNA libraries. Here, we provide direct evidence that unsp ecific priming events account for the majority of these rodent clones. Using an Alu consensus primer hn-RNA human specific c-DNA libraries h ave been established and the specificity of Alu-priming has been evalu ated. Using a variety of purification schemes for isolating hn-RNA we have significantly reduced the percentage of unspecific priming events . We also included a comparison of the hn-RNA yield from different som atic hybrids prior and after purification.