Quantitative DNA Fiber Mapping Research Articles

Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

Quantitative DNA fiber mapping

High resolution physical maps are indispensable for large-scale, cost-effective gene discovery. The construction of such maps of the human genome and model organisms is one of the major goals of the human genome project. The precise localization of cloned DNA fragments within much larger genomic fragments and knowledge of the extent of overlap between two clones are needed to assemble high resolution physical maps. The fluorescence in situ hybridization (FISH) provides this critical information. Isolation of DNA from cell nuclei and preparation of some sort of DNA “fibers,” that is, chromatin or 30-nm fibers representing bundles of DNA improves accessibility of the DNA target for probes and detection reagents and thus increases the hybridization efficiency. FISH applied to most preparations of decondensed nuclear or isolated cloned DNA allows visualization of probe overlap and provides some information of the existence and size of gaps between two clones. In quantitative DNA fiber mapping (QDFM), a solution of purified DNA molecules is placed on a flat surface prepared so that the DNA molecules slowly attach at one or both ends. Because of the high hybridization efficiency obtained with DNA fibers, QDFM is also the method of choice to optically map expressed sequences in limited genomic intervals.

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

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.

High-Resolution Physical Map of the Immunoglobulin λ Variant Gene Cluster Assembled by Quantitative DNA Fiber Mapping

Quantitative DNA fiber mapping (QDFM) allows rapid construction of near-kilobase-resolution physical maps by hybridizing specific probes to individual stretched DNA molecules. We evaluated the utility of QDFM for the large-scale physical mapping of a rather unstable, repeat-rich 850-kb region encompassing the immunoglobulin λ variant (IGLV) gene segments. We mapped a minimal tiling path composed of 32 cosmid clones to three partially overlapping yeast artificial chromosome (YAC) clones and determined the physical size of each clone, the extent of overlap between clones, and contig orientation, as well as the sizes of gaps between adjacent contigs. Regions of germline DNA for which we had no YAC coverage were characterized by cosmid to cosmid hybridizations. Compared to other methods commonly used for physical map assembly, QDFM is a rapid, versatile technique delivering unambiguous data necessary for map closure and preparation of sequence-ready minimal tiling paths.

Construction of two near-kilobase resolution restriction maps of the 5' regulatory region of the human apolipoprotein B gene by quantitative DNA fiber mapping (QDFM).

Quantitative DNA fiber mapping (QDFM) is a high-resolution technique for physical mapping of DNA. The method is based on hybridization of fluorescently labeled DNA probes to individual DNA molecules stretched on a chemically modified glass surface. We now demonstrate and validate a rapid QDFM-based approach for the mapping of multiple restriction sites and precise localization of restriction fragments in large genomic clones. Restriction fragments of a 70-kb P1 clone (P1-70) containing the 5' region of the human apolipo-protein B gene (APOB) were subcloned and mapped along straightened P1-70 DNA molecules. Multicolor fluorescence in situ hybridization (FISH) and digital image analysis allowed us to rapidly position 29 restriction fragments, ranging in size from 0.5 kb to 8 kb, and to map 43 restriction sites. The restriction map obtained by QDFM was in excellent agreement with information obtained by RecA-assisted restriction endonuclease (RARE) cleavage, long-range PCR, and DNA sequence analyses of the P1-70 clone. These data demonstrate that QDFM is a rapid, reliable method for detailed restriction site-mapping of large DNA clones.

High sensitivity, high resolution physical mapping by fluorescence in situ hybridization on to individual straightened DNA molecules

High resolution physical mapping of clonal DNA fragments with kilobase (kb) resolution can now be performed rapidly by fluorescence in situ hybridization (FISH) onto individual DNA molecules (DNA fibers). We developed a sensitive procedure termed 'quantitative DNA fiber mapping' which consists of three steps: preparation of DNA fibers, hybridization of non-isotopically labeled probes and determination of the relative mapping position by fluorescence image analysis. The DNA fibers are produced by binding linearized DNA molecules with one or both ends to a solid substrate followed by homogeneous stretching of the molecule by the action of a receding meniscus during drying ('molecular combing'). In a slight variation of this protocol, we deposit circular DNA molecules. Substrates for DNA immobilization are glass slides, coverslips or thin sheets of mica derivatized with amino-silane. Probes are prepared to counterstain the DNA fibers, from the clones to be mapped and for specific landmarks along linear or circular DNA molecules such as cloning vector sequences. Following hybridization and immunocytochemical detection of bound probes, images are analysed and relative distances are recorded for map assembly. Here, we describe our experience with substrate preparation, molecular combing and mapping of cloned or enzymatically synthesized probes ranging in size from 1.2 kb to 100 kb along DNA molecules that are between 17 kb and 1200 kb in size.

Quantitative DNA fiber mapping.

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 approximately 2.3 kb/microns 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 approximately 49 kb long lambda phage DNA molecules with approximately 1 kb precision; (ii) localization of the overlap between a cosmid and a colinear P1 clone; and (iii) mapping of P1 clones along an approximately 490 kb yeast artificial chromosome (YAC) with approximately 5 kb precision and estimation of the approximately 25 kb gap between them.