Libraries for each human chromosome, constructed from sorter-enriched chromosomes by using linker-adaptor PCR.
We describe here the production of complex libraries enriched in sequences from each human chromosome type, starting with only a few thousand sorter-purified chromosomes. In this procedure, DNA is extracted from the sorted chromosomes, digested to completion by using the frequently cutting restriction endonuclease Sau3A1, and ligated, on each end, to an adaptor oligonucleotide. These fragments are then amplified using PCR with a sequence homologous to the adaptor oligonucleotide as a primer. We have used this procedure to produce PCR libraries for each of the 24 human chromosomes. These libraries were characterized by gel electrophoresis and found to be composed of a continuum of sequences ranging in size from a few hundred to approximately 1,000 bp. The libraries, when used as probes for fluorescence in situ hybridization, stained the target chromosomes more or less continuously, even after PCR amplification for more than 200 cycles. These libraries are useful as hybridization probes to facilitate molecular cytogenetic studies and as sources of probes either for identification of polymorphic short tandemly repeated sequences or for development of sequence-tagged sites.
Vooijs
M
M
Department of Laboratory Medicine, University of California, San Francisco 94143-0808.
Yu
L C
LC
Tkachuk
D
D
Pinkel
D
D
Johnson
D
D
Gray
J W
JW
D001483:Base Sequence; D002460:Cell Line; D002877:Chromosomes, Human; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015342:DNA Probes; D015723:Gene Library; D008040:Genetic Linkage; D006801:Humans; D007621:Karyotyping; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping
D015342:DNA Probes; D009838:Oligodeoxyribonucleotides; D004247:DNA