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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.

Original publication

DOI

10.1016/0888-7543(90)90548-9

Type

Journal article

Journal

Genomics

Publication Date

06/1990

Volume

7

Pages

257 - 263

Keywords

Animals, Base Sequence, Chromosomes, Human, Cloning, Molecular, Cricetinae, DNA Probes, Humans, Hybrid Cells, Molecular Sequence Data, Polymerase Chain Reaction, Repetitive Sequences, Nucleic Acid