The assembly of sequence ready, high-resolution physical maps and construction of minimally overlapping contigs for the human as well as model genomes requires accurate determination of the extent of overlap between adjacent clones as well as their relative orientation. This is presently done by procedures such as clone fingerprinting, Southern blot analysis or clone end sequencing. We present a complementary analytical technique to map directly cloned DNA sequences on to individual stretched DNA molecules. This approach uses the hydrodynamic force of a receding meniscus to prepare straight high molecular weight DNA molecules that provide a linear template of ∼2.3 kb/μm on to which the cloned probes can be mapped by in situ hybridization. This technique has numerous advantages such as a very high density of mapping templates, reproducible stretching of the mapping template providing a linear genomic scale, determination of clone orientation and direct visualization of DNA repeats. The utility and accuracy of quantitative DNA fiber mapping are illustrated through three examples: (i) mapping of lambda DNA restriction fragments along linearized ∼49 kb long lambda phage DNA molecules with ∼1 kb precision; (ii) localization of the overlap between a cosmid and a colinear P1 clone; and (iii) mapping of P1 clones along a ∼490 kb yeast artificial chromosome (YAC) with ∼5 kb precision and estimation of the ∼25 kb gap between them.
ASJC Scopus subject areas
- Molecular Biology