Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms

Abstract

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable. I dentifying polymorphic sites in the genome is a basic aspect of molecular genetics and genomics. The process is needed for a variety of purposes, ranging from the development of polymorphic marker sets useful as a tool for genetic analysis of a chromosomal region or full genome scan, to the initial identification of variants or mutations in a newly discovered gene (1, 2). In most cases, the identity of base differences and their location relative to a gene or other polymorphic sites is either useful or required. Recent estimates of the number of singlenucleotide polymorphisms (SNPs) needed for whole genome association studies in humans vary from several thousand to several hundred thousand (1, 3); thus, efficient and cost-effective methods for identifying a large number of SNPs with the required characteristics of dense yet even spacing, and of known order over large uncharacterized regions of the genome, is of interest. A comparison of two methods to develop a densely ordered map of SNPs covering a 4-Mb region of the human genome was recently reported (4). In one approach, large-insert bacterial clones, bacterial artificial chromosomes (BACs) (5) and P1 artificial chromosomes (PACs) (6), spanning this region were fragmented and reconstructed in 2-kb plasmid libraries, which were then sequenced. This shotgun procedure is efficient in identifying SNPs; however, to approach a map of 30-kb average SNP spacing, bidirectional sequencing of approximately 500 randomly chosen subclones per 100 kb of genomic sequence was required. Multiple BAC and PAC clones mapping to the region...

Trapping Enhancers by Transgenic Expression of BACs Engineered in Bacteria with loxP Transposons

“…It does not require sequence homology between vector and genomic inserts of BACs to introduce exogenous DNA cassettes. Insertions of the bacterial transposon Tn10 can introduce exogenous DNA, including lox sites, at random locations in BACs [56,57]. Furthermore, site-specific recombination by the Cre-lox system can deliver these reporter genes and other exogenous DNA precisely to the ends of genomic DNA inserts in BACs [58][59][60].…”

“…The DNA in BAC clones is isolated by simple mini-prep procedures, digested with Not I enzyme and analyzed by FIGE [63,64]. High throughput formats for preparing DNA from BAC deletions in 96-well plates, suitable for subsequent FIGE analyses and end sequencing, have also been described [57,64].…”

Long range gene-regulatory sequences identified by transgenic expression of bacterially-engineered enhancer-trap BACs in zebrafish

“…Germ-line expression of the modified BAC in zebrafish or mice has been achieved with yet a third recombination system, namely the vertebrate Tol2 system, described later in the article. Insertions of the bacterial transposon Tn10 can introduce exogenous DNA, including lox sites, at random locations in BACs [39,40]. Site-specific recombination by the Cre-lox system on the other hand, can deliver these reporter genes and other exogenous DNA precisely at the ends of genomic DNA inserts in BACs [41][42][43].…”

“…Prior to the availability of whole genome sequences it offered probably the best means to unambiguously map genetic markers at the sequence level. Thus polymorphisms have been mapped on physical maps of chromosomes using the technology [40,41]. Long-range regulatory elements of the mouse Nkx2-5 gene have also been mapped functionally using end-deleted EGFP functionalized BACs to generate transgenic mice [63].…”

“…Note that orientation of arrow refers to directionality of loxP sequence. This feature has been utilized to place numerous DNA cassettes in the BAC, such as: i) mammalian cell-selectable antibiotic resistance genes [41], ii) a basal promoter-containing EGFP gene to serve as an enhancer-trap to functionally locate potential generegulatory elements further upstream [40], and iii) introduce end sequences of the vertebrate transposn iTol2 to generate integration-ready GFP-functionalized BAC DNA for zebrafish or mouse transgenesis studies [43,62].…”