Applications of Quantitative DNA Fiber Mapping (QDFM) in Physical Mapping And Sequencing*

Heinz-Ulrich G. Weier, Mei Wang, Jan-Fang Cheng, Yiwen Zhu, Herbert W. Moise, Christopher H. Martin, Micheal J. Palazzolo and Joe W. Gray

Resource for Molecular Cytogenetics and Human Genome Center, Life Sciences Division, 74-157, University of California, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.

"Quantitative DNA Fiber Mapping" (QDFM) recently developed in our laboratory combines three techniques (MOLECULAR COMBING, FISH and IMAGE ANALYSIS) for precision mapping of individual DNA fragments and determination of distance or overlap between pairs of cloned DNA fragments.

In MOLECULAR COMBING, a solution of purified DNA molecules is placed on a silanated glass slide prepared so that the DNA molecules slowly attach at one or both ends. The DNA solution is then spread over a larger area by placing a coverslip on top, DNA molecules are allowed to bind to the surface and dried. Individual DNA molecules are straightened and uniformly stretched during drying by the hydrodynamic action of the receding meniscus. The position of specific sequences along the stretched DNA molecules is visualized by overnight fluorescence in situ hybridization (FISH) and measured by digital IMAGE ANALYSIS techniques on images recorded from the fluorescence microscope.

In pilot experiments, we applied QDFM to map gamma alpha transposons, plasmid or cosmid probes along P1 molecules, and P1 or PAC DNA clones along straightened YAC molecules ranging in size from ~490kb to >1Mbp[1]. Our studies demonstrated the power of QDFM by showing that:

1. molecular combing and high resolution physical mapping can be performed on DNA molecules linearized by digestion with restriction enzymes, randomly broken DNA or circular molecules,

2. linear DNA molecules ranging in size range from 17kb to >1Mbp are uniformly stretched to ~2.3kb/µm so that measurements obtained by image analysis [measured in µm] can be converted directly to genomic distances [measured in kb],

3. only few (<10) molecules are needed for analysis,

4. the hybridization efficiency is high so that DNA fragments of less than 1kb can be mapped,

5. plasmids (such as ~3kb sequencing templates), mobilized transposons and cosmid probes can be mapped to within ~2-5 kb along P1 molecules of ~55-95 kb,

6. the extent and orientation of overlap between two P1 DNA molecules can be determined to within ~3 kb by hybridizing DNA from one clone onto linearized molecules of the other,

7. the map position of two independent P1 clones along a YAC molecule can be measured with a resolution of a few kb,

8. the physical distance between two P1 clones representing the proximal ends of two contigs (gap region) can be measured with kilobase resolutions and

9. the chimerism status of sequencing templates can be determined rapidly.

The impact of QDFM on genome research will depend on how well it scales-up to accommodate the needs of large-scale mapping and sequencing projects. Preliminary results showed that as many as 20 clones can be combed on a single microscope slide. Furthermore, QDFM is highly amenable to automation, which might increase its throughput by orders of magnitude. Molecular combing and FISH require only little user interaction and instrumentation for slide handling (washes, staining etc.) exists. Development of semi-automated slide scanning and image acquisition/analysis should facilitate these aspects of the analysis procedure. These developments should bring QDFM to the point where it is of major utility in assembly of sequence-ready physical maps and quality control during the sequencing process.

* Supported by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research, Department of Energy, under contract DE-AC-03-76SF00098.

1 H. -U. G. Weier et al. Human Molecular Genetics 4.1903-1910 (1995).