GENERATION OF A CHROMOSOME-22-SPECIFIC C-DNA LIBRARY AS CONFIRMED BY FISH ANALYSIS

We have recently developed a strategy for the rapid enrichment of c-DN A fragments from selected human chromosomes. Heteronuclear RNA (hn-RNA ) is isolated from a somatic cell hybrid that retains a single human c hromosome in a rodent background. Following c DNA synthesis, human seq uences are selectively amplified by the Alu polymerase chain reaction (Alu-PCR). Here we have applied this protocol for the selective isolat ion of novel c-DNAs encoded by chromosome 22. Fluorescence in situ hyb ridization has been used to confirm the chromosome-22-specific origin of the c-DNA fragments. Controls show DNAse-free RNase-treated hn-RNA results in no c-DNAs or Alu-PCR products. As demonstrated by competiti ve in situ suppression hybridization (CISS), the majority of the Alu-P CR products from hybrid GM 10027 are located on chromosome 22. Without competition, hybridization signals have also been identified on other human chromosomes. These unspecific hybridization signals result from Alu sequences and can successfully be reduced by competition with cot 1 DNA. This is the first report of the use of CISS for the localizati on of chromosome-specific c-DNAs.