Cross-screening: a new method to assemble clones rapidly and unambiguously into contigs.

Abstract

We have developed a new procedure that relies on an array of cross-hybridization tests to order a set of random clones into a contig. The method, called cross-screening, uses each clone as a target and its end sequences as probes, in a matrix of reciprocal cross-hybridization tests performed on a single blot. The relationships among the clones are determined rapidly from the pairwise tests, allowing clone order to be determined directly. We have applied this technique to DNAs from a set of overlapping ~ clones from Drosophila chromosome 4. The location and orientation of each clone derived from the cross-screening data was that expected from the map assembled from overlapping restriction sites and chromosomal walking. The procedure provided additional information on a previously unknown, internally repeated DNA sequence. To demonstrate the general utility of the procedure, we have applied it to a previously described clone set within a contig in region 22q12 of human chromosome 22. The correct relative position and orientation of these clones were derived from the cross-screening data without knowledge of, or reference to, any nucleotide sequence or restriction site analysis of the DNA concerned. The cross-screening procedure is fast, economical, and robust and allows clone overlaps to be determined efficiently, with minimal interference from repeated DNA sequences. This new procedure is specifically designed for small groups of overlapping clones [tens to hundreds] and should facilitate the ordering of subclone libraries derived from small chromosomes or the large cloned inserts carried in YAC, BAC, and PI vectors.

The fourth chromosome of Drosophila melanogaster : Interspersed euchromatic and heterochromatic domains

“…The cytological positions of the transgene inserts were determined with polytene chromosomes isolated from late third instar larvae (24) hybridized with the P element construct as a probe (10). Map positions were established at higher resolution by hybridization of cloned fragments of adjacent genomic DNA to cosmid and bacterial artificial chromosome (BAC) clones from the fourth chromosome (20,45). The DNA flanking the P element inserts was cloned in vector pCR3.1 (Invitrogen) by using inverse PCR (14).…”

“…Genetic analysis indicated that line 2-M1021R is an allele of ci; homozygotes have a fusion of wing veins 4 and 5 because of inappropriate expression of ci (R. Holmgren, personal communication). Sequencing of the tag clone places the insertion site within a cluster of repeats upstream of the ci transcript (20). This 3-kilobase region has a similar, nonidentical, AT-rich sequence present approximately every 200 bp.…”

“…20). Genetic analysis indicated that line 2-M1021R is an allele of ci; homozygotes have a fusion of wing veins 4 and 5 because of inappropriate expression of ci (R. Holmgren, personal communication).…”

“…However, the resolution that can be obtained with this approach is limited. To generate a higher resolution map, providing an unambiguous order for the insertion sites, the flanking DNA for each transgene was cloned by inverse PCR (14); these ''tag clones'' were used to screen an ordered collection of cosmid and BAC clones from the fourth chromosome (20,45). By assigning each tag clone to one or more cosmid and͞or BAC clones (Table 1), we have been able to derive the relative positions of the transgene insertion sites for the R and V lines (Fig.…”

“…Most of the chromosome, 3-4 megabases, is comprised of simple satellite repeats and does not contain any known genes. The remaining portion (estimated at 1.2 megabases) has a banded pattern in polytene chromosomes (cytological region 101E-102F; Ϸ1% of the total bands); it appears to be a mosaic of middle and low copy repetitive DNA (often transposons) interspersed with unique DNA (17)(18)(19)(20). Immunofluorescent staining with antibodies against HP1 gives a banded pattern across this region (21).…”