Enrichment of chromosome specific hncDNAs by magnetic bead coupled Alu sequences.
We have employed a strategy for the rapid enrichment of cDNA clones from human chromosome 22 utilizing magnetic beads. Starting from a somatic cell hybrid which retains chromosome 22 in rodent background, heteronuclear (hn) RNA was transcribed into hncDNA using poly dT-primers. Using linker specific primers hncDNA was amplified by PCR. To identify chromosome 22 specific hncDNAs a highly human specific Alu consensus sequence (PD39) was biotinylated and hybridized to the PCR product of the hncDNAs in solution. Hybridized hncDNA-PD39 complexes are captured using streptavidin-coated magnetic beads. Hybridized hncDNAs are selectively amplified by PCR. To verify the chromosome specificity the hncDNA was used as probe for in situ hybridization. Following two rounds of selection with magnetic beads there was an increasingly strong hybridization signal on chromosome 22. The capturing of hncDNAs by magnetic beads as described in this study is faster and more efficient than previously described methods for the isolation of chromosome specific hncDNAs. The novel approach has been employed to generate hncDNAs highly enriched for chromosome 22 specific sequences.
Overmyer
K
K
Department of Human Genetics, Medical School, University of Saarland, Homburg/Saar, Germany.
Müller
H W
HW
Gimbel
W
W
Göttert
E
E
Meese
E
E
D000818:Animals; D001483:Base Sequence; D002892:Chromosomes, Human, Pair 22 / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D008280:Magnetics; D012091:Repetitive Sequences, Nucleic Acid
D017931:DNA Primers; D018076:DNA, Complementary