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An efficient method using short oligonucleotides is described for the isolation of minisatellite markers of the type variable number of tandem repeats (VNTR). It is used to screen 350 cosmids from chromosome 7 for the presence of such potentially polymorphic DNA segments. From the number of hybridization signals to chromosome 7 cosmids, I estimate the human genome to have at least about 15000 VNTR loci containing minisatellite core sequences, which is well above previous estimates.

A cosmid library has been constructed from a hamster x human hybrid cell line and gridded into 270 microtiter plates containing a total of 25,920 single colonies. Approximately 84% of the recombinants contain human material, with an average length of 29 kb. This library represents a nearly three-fold coverage of human chromosome 4. We investigated this library for presumptive genes, using a set of oligonucleotides detecting SpI and splice-site consensus sequences. The presence of simple repeat motifs was investigated in the cosmids using the oligonucleotides (GGATTT)3, (GGAT)4, (CAC)5, (GCC)5, (AGC)5, (GATA)4, (GACA)4, and (CA)8 as hybridization probes.

To facilitate the identification of genes within genomic DNA, we have developed a method based on the use of short oligonucleotides designed from the consensus sequences of splice sites. We describe here the hybridization and washing conditions under which such oligonucleotides can be used to screen cosmid libraries. We confirm the presence of genes within cosmids identified by screening with one oligonucleotide by showing that DNA isolated from such cosmids will hybridize to another splice-site oligonucleotide.

We have developed a method to identify clones containing recognition sequences for enzymes that cut mammalian genomes infrequently by direct screening of genomic libraries. The degenerate oligonucleotide NNGCGGCCGCNN, in which the internal 8 bases correspond to the recognition sequence of Not I, was used to screen a cosmid library, and it led to a greater than 10-fold enrichment in the number of clones containing Not I sites. This technique permits the efficient identification of sufficient clones from a chromosome-specific library to allow the construction of a complete pulsed-field map of that chromosome and to assist in finding genes in genomic DNA.