Rational design of landmark probes for quantitative DNA fiber mapping (QDFM)

Abstract

Rapid construction of high-resolution physical maps requires accurate information about overlap between DNA clones and the size of gaps between clones or clone contigs. We recently developed a procedure termed 'quantitative DNA fiber mapping' (QDFM) to help construct physical maps by measuring the overlap between clones or the physical distance between non-overlapping contigs. QDFM is based on hybridization of non-isotopically labeled probes onto DNA molecules that were bound to a solid support and stretched homogeneously to approximately 2.3 kb/microm. In this paper, we describe the design of probes that bind specifically to the cloning vector of DNA recombinants to facilitate physical mapping. Probes described here delineate the most frequently used cloning vectors such as BACs, P1s, PACs and YACs. As demonstrated in representative hybridizations, vector-specific probes provide valuable information about molecule integrity, insert size and orientation as well as localization of hybridization domains relative to specifically-marked vector sequences.

Figure 1

(A–C) BAC clone #131 mapped to BAC clone #97. (A) A linear DNA fiber of BAC #97 fiber (blue), BAC #131 hybridization domain (red) and the PCR-synthesized vector-specific probes hybridization signals (green/red). (B) A schematic illustration of the different hybridization domains for the linear BAC molecule. (C) A circular molecule from the same hybridization mixture with all the hybridization domains as explained in (B). (D) A plasmid clone hybridized to a P1 clone (#41, blue). The hybridization shows two domains (red) indicating chimerism of the clone. The vector-specific probes (green/red) appeared on the lower left corner of the picture. (Insert) A comparison of hybridization signals recorded from either randomly broken linear or circular DNA molecules. (E–G) Mapping of a BAC clone 97A23 onto a PAC clone 11C11. (E) A circular PAC clone 11C11 DNA fiber (blue) was hybridized with BAC clone 97A23 (red) and PAC vector-specific probes (green/red). (F) A schematic drawing of each hybridization domain and the extent of overlap. (G) A schematic of hybridization domains. The PCR synthesized vector-specific probe (red) is located near the T7 end of the vector pAd10SacBII (green). (H–I) Hybridization of two P1 clones #1107 and #1143 onto YAC clone 141G6. (H) Probes of two P1 clones #1107 and #1143 (red) and the pYAC3 vector (red) were hybridized onto a YAC clone 141G6 DNA fiber (green). The hybridization of the vector probe pYAC3 (red dots at both ends) indicates a complete YAC DNA fiber. (I) A schematic drawing showing the extent of the hybridization domains and the pYAC3 vector probe signals (red) on both ends.