Rapid isolation of DNA probes within specific chromosome regions by interspersed repetitive sequence polymerase chain reaction.
A method was recently developed for the specific amplification of human DNA sequences from interspecific somatic cell hybrids by the polymerase chain reaction (PCR) using primers directed to Alu, a short interspersed repeat element (SINE). We now show human-specific amplification using a primer to the 3' end of the human long interspersed repeat element L1Hs (LINE). A monochromosomal hybrid containing an intact human X chromosome yielded approximately 25 discrete products, ranging in size from 800 to 4500 bp. Combination of a single Alu primer and the L1Hs primer yielded a large number of smaller products (300-1000 bp) distinct from those observed with either primer alone. Inspection of ethidium bromide-stained gels showed one Alu-Alu and three Alu-L1Hs products which were present in an intact X chromosome but absent in a hybrid containing an X chromosome deleted for the single metaphase band q28. These four fragments were isolated from the gel and used as probes on Southern blots which confirmed their localization to Xq28. These results demonstrate that primers can be constructed to a variety of interspersed repetitive sequences (IRS) and used individually or in combination for the rapid isolation of DNA fragments from defined chromosomal regions by IRS-PCR.
Ledbetter
S A
SA
Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030.
Nelson
D L
DL
Warren
S T
ST
Ledbetter
D H
DH
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002874:Chromosome Mapping; D006224:Cricetinae; D003412:Cricetulus; D015342:DNA Probes / Q000302:isolation & purification*; D005784:Gene Amplification; D006801:Humans; D006822:Hybrid Cells / Q000032:analysis*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D014960:X Chromosome
D015342:DNA Probes