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-DNA fragments from selected human chromosomes. Heteronuclear RNA (hn-RNA) is isolated from a somatic cell hybrid that retains a single human chromosome in a rodent background. Following c-DNA synthesis, human sequences are selectively amplified by the Alu polymerase chain reaction (Alu-PCR). Here we have applied this protocol for the selective isolation of novel c-DNAs encoded by chromosome 22. Fluorescence in situ hybridization 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 competitive in situ suppression hybridization (CISS), the majority of the Alu-PCR 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 localization of chromosome-specific c-DNAs.
Göttert
E
E
Institut für Humangenetik, Universität des Saarlandes, Homburg/Starr, Germany.
Klein
V
V
Piontek
K
K
Overmyer
K
K
Zang
K D
KD
Meese
E
E
D000818:Animals; D001667:Binding, Competitive; D002892:Chromosomes, Human, Pair 22; D004247:DNA / Q000032:analysis*; D015723:Gene Library; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016133:Polymerase Chain Reaction
D004247:DNA