Isolation of DNA fragments from a human chromosomal subregion by Alu PCR differential hybridization.
The recent advent of Alu element-mediated PCR (Alu PCR) allows the rapid isolation of human-specific fragments from mixed DNA sources. This technique greatly facilitates the isolation of DNA fragments from specific regions of the human genome. We report a novel technique utilizing Alu PCR products as differential hybridization probes to isolate human DNA fragments from a chromosomal subregion. We used the Alu PCR products from a pair of somatic cell hybrids in which the human DNA content differs only in the 5q11.2-q13.3 region as differential hybridization probes. One hybrid (GM10114) retains an intact chromosome 5, while the other (HHW1064) contains a chromosome 5 deleted for the q11.2-q13.3 region. Phage from a flow-sorted chromosome 5 library were hybridized with the Alu PCR synthesis product from the chromosome 5 hybrid. Positively hybridizing phage were then screened with the Alu PCR product from the deletion 5 hybrid. Phage that hybridized to the Alu PCR product of the chromosome 5 hybrid but did not hybridize to the Alu PCR product of the deletion 5 hybrid were further characterized. We isolated five phage from 5q11.2-q13.3 using this differential hybridization procedure. Only one of these phage corresponded to a detectable difference between the ethidium bromide-stained Alu PCR products of the two somatic cell hybrids. This technique should be applicable to any somatic cell hybrid-deletion hybrid pair.
Bernard
L E
LE
Department of Medical Genetics, University of British Columbia, Vancouver, Canada.
Brooks-Wilson
A R
AR
Wood
S
S
D000818:Animals; D001435:Bacteriophages / Q000302:isolation & purification; D001483:Base Sequence; D015139:Blotting, Southern; D002872:Chromosome Deletion; D002895:Chromosomes, Human, Pair 5; D006224:Cricetinae; D004247:DNA / Q000302:isolation & purification*; D015342:DNA Probes; D006801:Humans; D006822:Hybrid Cells; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid
D015342:DNA Probes; D004247:DNA