The construction of human somatic cell hybrids containing portions of the mouse X chromosome and their use to generate DNA probes via interspersed repetitive sequence polymerase chain reaction.
Interspersed repetitive sequence polymerase chain reaction (IRS-PCR) has become a powerful tool for the rapid generation of DNA probes from human chromosomes present in rodent somatic cell hybrids. We have constructed a somatic cell hybrid containing a major portion of the mouse X chromosome in a human background (clone 8.0). IRS-PCR was developed for the specific amplification of mouse DNA using either of two primers from the rodent-specific portion of the murine B1 repeat. Amplification was subsequently performed with clone 8.0 and a subclone, 8.1/1, which retains a small murine X-chromosomal fragment including Hprt and the Gdx locus. A total of 15-20 discrete PCR products ranging in size from less than 500 to greater than 3000 bp were obtained from clone 8.0 with each primer. In clone 8.1/1, a subset of these bands plus some additional bands were observed. Nine bands amplified from clone 8.1/1 have been excised from gels and used as probes on Southern blots. All of the fragments behaved as single-copy probes and detected domesticus/spretus variation. They have been regionally mapped using an interspecific backcross. The probe locations are compatible with those of markers known to be present in clone 8.1/1. These results demonstrate the feasibility of this method as applied to the mouse genome and the high likelihood of generating useful DNA probes from a targeted region.
Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030.
G E
GE
Herman
M
M
Berry
E
E
Munro
I W
IW
Craig
E R
ER
Levy
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular / Q000379:methods; D015342:DNA Probes; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology / Q000502:physiology; D007621:Karyotyping; D007739:L Cells / Q000166:cytology / Q000502:physiology; D008677:Metaphase; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014960:X Chromosome
D015342:DNA Probes; D009838:Oligodeoxyribonucleotides