Rapid isolation of human chromosome-specific DNA probes from a somatic cell hybrid.
A strategy for the rapid isolation from rodent hybrids of human chromosome-specific probes by enzymatic amplification is described. Synthetic oligonucleotide primers based on the consensus Alu sequence were used to amplify inter-Alu sequence from total human genomic DNA and from a somatic cell hybrid, PNTS-1, containing one homolog of chromosome 5 as its only human complement. Direct sequence analysis of the products from human genomic DNA confirmed their inter-Alu structure and provided a novel means for the examination of the 5' end of the Alu consensus. The amplified sequences from the somatic cell hybrid DNA were cloned into a plasmid vector by blunt-end ligation, yielding clones with inserts in the range 300 to 1000 bp. More than 80% of these clones carried inserts that behaved essentially as single-copy human sequences. Hybridization of a selection of these clones to human DNA, hamster DNA, and the original hybrid DNA confirmed that they were derived from chromosome 5. Direct sequence analysis of the vector/insert boundaries in two clones confirmed that inter-Alu sequences had been cloned. This approach has significant advantages over other methods of isolating chromosome-specific probes from hybrid cells, enabling direct separation and cloning of human DNA probes that can be readily used for mapping studies.
Cotter
F E
FE
ICRF Medical Oncology Laboratory, St. Bartholomew's Hospital, London, United Kingdom.
Hampton
G M
GM
Nasipuri
S
S
Bodmer
W F
WF
Young
B D
BD
D000818:Animals; D001483:Base Sequence; D002877:Chromosomes, Human; D003001:Cloning, Molecular; D006224:Cricetinae; D015342:DNA Probes / Q000302:isolation & purification*; D006801:Humans; D006822:Hybrid Cells; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid
D015342:DNA Probes