Optical mapping of lambda bacteriophage clones using restriction endonucleases

Optical mapping is an emerging single molecule approach for the rapid generation of ordered restriction maps, using fluorescence microscopy. We have improved the size resolution of optical mapping by imaging individual DNA molecules elongated and fixed onto derivatized glass surfaces. Averaged fluorescence intensity and apparent length measurements accurately determined the mass of restriction fragments 800 basepairs long. We have used optical mapping to create ordered restriction maps for lambda clones derived from the mouse pygmy locus.     

Optical mapping of DNA polymerase I action and products

Single molecule approaches to the characterization of biochemical systems offer an intrinsically simple and direct approach to address difficult, previously unyielding problems. Optically based approaches have recently been used to construct high resolution, ordered restriction maps from a variety of clone types. Advancements in surface technologies have enabled the reliable elongation and fixation of large DNA molecules onto specially derivatized substrates with retention of biochemical accessibility. In this study, the addition of fluorescently labeled nucleotides to surface-mounted DNA molecules by the action of DNA polymerase I is investigated using fluorescence microscopy to image individual template molecules. Molecules undergoing nick translation and containing only a few fluorochromes are readily imaged. These novel results suggest that surface-bound molecules may serve as a substrate for a broad range of enzymatic actions, and may offer new routes to analysis when coupled to advanced imaging techniques.     

Scanning force microscopy of DNA molecules elongated by convective fluid flow in an evaporating droplet

Scanning force microscopy (SFM) was used to image intact, nearly fully elongated lambda bacteriophage DNA molecules, fixed onto freshly cleaved mica surfaces. Molecular elongation and fixation were accomplished using a newly characterized fixation technique, termed "fluid fixation." Here convective fluid flows generated within an evaporating droplet of DNA solution efficiently elongate DNA molecules for fixation onto suitably charged surfaces. SFM images of a very large bacteriophage genome, G, showed the presence of double-stranded bubbles. We speculate that these structures may contain putative replication forks. Overall, the experiments presented here demonstrate the viability of using fluid fixation for the preparation of DNA molecules for SFM imaging. The combination of largely automatable optically based techniques with the high-resolution SFM imaging presented here will likely produce a high-throughput system for detailed physical mapping of genomic DNA or clones.     

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.     

Construction and validation of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease cleavage

RecA-assisted restriction endonuclease (RARE) cleavage is an "Achilles' heel" approach to restriction mapping whereby a RecA-protein-oligodeoxynucleotide complex protects an individual restriction site from methylation, thus limiting subsequent digestion to a single, predetermined site. We have used RARE cleavage to cut yeast artificial chromosomes (YACs) at specific EcoRI sites located within or adjacent to sequence-tagged sites (STSs). Each cleavage reaction produces two YAC fragments whose sizes are a direct measure of the position of the STS in the YAC. In this fashion, we have positioned 45 STSs within a contig of 19 independent YACs and constructed a detailed RARE-cleavage map that represents 8.4 Mbp of human chromosome 6p21.3-22. By comparing maps of overlapping YACs, we were able to detect seven internal deletions that ranged from approximately 75 kbp to approximately 1 Mbp in size. Thirteen pairs of EcoRI sites were targeted for double RARE cleavage in uncloned total human DNA. The excised fragments, up to 2 Mbp in size, were resolved by pulsed-field gel electrophoresis and were detected by hybridization. In general, the genomic RARE-cleavage results support the YAC-based map. In one case, the distance in uncloned DNA between the two terminal EcoRI sites of a YAC insert was approximately 1 Mbp larger than the YAC itself, indicating a major deletion. The general concept of RARE-cleavage mapping as well as its applications and limitations are discussed.     

Restriction map of a 35-kb HLA fragment constructed by nested deletion 'drop-out' mapping

An efficient method for generating detailed restriction maps of large cloned DNA segments is demonstrated. The mapping strategy entails comparing restriction fragments from a parent clone and from nested deletion derivatives of that clone. In a set of deletion plasmids of decreasing size, an individual fragment will be lost, or 'drop-out', according to its position in the cloned fragment. In this demonstration, nested deletions were generated in both directions in a 35-kb DNA segment from the human leukocyte antigen (HLA) region by intramolecular transposition of an engineered gamma delta (Tn1000) element present in a special 'deletion factory' cloning vector [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 7874-7878]. Fifteen plasmids with deletions extending in one direction and eleven plasmids with deletions extending in the opposite direction were digested singly by each of four restriction enzymes. A total of 36 cleavage sites were mapped in the 35-kb HLA fragment. This drop-out approach using nested deletions provides a simple and efficient means of mapping restriction sites, genes and other features of interest in cosmid-sized cloned DNA segments or DNAs.     

A NotI restriction map of the entire long arm of human chromosome 21

A variety of maps of the human genome have been constructed, including cloned DNA maps. We have isolated 40 of the 42 NotI sites that exist on the long arm of human chromosome 21, as NotI linking clones and constructed a complete NotI restriction map spanning the entire region. This map, which provides the most reliable ordering and distance estimation in the region from a pericentromeric locus to the terminus, demonstrates the usefulness of linking clone mapping for analysing human chromosomes.     

A P1-based physical map of the region from D17S776 to D17S78 containing the breast cancer susceptibility gene BRCA1

BRCA1, a breast and ovarian cancer susceptibility locus, has been isolated and maps to 17q21. A physical map of the BRCA1 region which extended from the proximal boundary at D17S776 to the distal boundary at D17S78 was constructed and consists of 51 sequence tagged sites (STSs) from P1 and YAC ends, nine new short-tandem repeat (STR) polymorphic markers, and eight identified genes. The contig, which spans the estimated 2.3 Mb region, contains 29 P1s, 11 YACs, two BACs, and one cosmid. Based on key recombinants in two linked families, BRCA1 was further localized to a region bounded by D17S1321 on the proximal side and D17S1325 on the distal side. Within this estimated 600 kb region, the contig was composed completely of P1s and BACs ordered by STS-content mapping and confirmed by DNA restriction fragment fingerprinting.     

Cleavage of adeno-associated virus DNA with Sali,Psti and Haeii restriction endonucleases

Duplex AAV-2 DNA was digested with SalI, PstI or HaeII restriction endonucleases and the cleavage sites were mapped. SalI cleaves AAV DNA at 0.310 map units, PstI at 0.106, 0.422 and 0.914 and the five HaeII sites were mapped at 0.110. 0.156, 0.181, 0.536 and 0.600 map units. These cleavage products will be useful for the isolation of specific regions from the AAV DNA, located outside of the stably transcribed region of the genome, and will also help to map more complex restriction enzyme cleavages.     

A nonradioactive method for improved restriction analysis and fingerprinting of large P1 artificial chromosome clones

The P1 artificial chromosome (PAC) cloning system is very useful for physical mapping, however, the large insert sizes cause difficulty in routine restriction analysis. In order to facilitate restriction mapping and fingerprinting, we have developed a simple, nonradioactive method for end-labeling and detection of restriction fragments from PAC clones. This method is very easy to implement, gives good differentiation of restriction fragments, and uses comparatively small amounts of DNA. We have used this method for restriction analysis of PAC clones containing inserts from human as well as from lower vertebrates. The method should also be applicable to other large-insert plasmid systems.     

Long-term impact of reproductive factors on cancer risk

In the program ODS we provide a methodology for quickly ordering random clones into a physical map. The process of ordering individual clones with respect to their position along a chromosome is based on the similarity of binary signatures assigned to each clone. This binary signature is obtained by hybridizing each clone to a panel of oligonucleotide probes. By using the fact that the amount of overlap between any two clones is reflected in the similarity of their binary signatures, it is possible to reconstruct a chromosome by minimizing the sum of linking distances between an ordered sequence of clones. Unlike other programs for physical mapping, ODS is very general in the types of data that can be utilized for chromosome reconstruction. Any trait that can be scored in a presence--absence manner, such as hybridized synthetic oligonucleotides, restriction endonuclease recognition sites or single copy landmarks, can be used for analysis. Furthermore, the computational requirements for the construction of large physical maps can be measured in a matter of hours on work-stations such as the VAX2000.     

Error checking and graphical representation of multiple-complete-digest (MCD) restriction-fragment maps

Genetic and physical maps display the relative positions of objects or markers occurring within a target DNA molecule. In constructing maps, the primary objective is to determine the ordering of these objects. A further objective is to assign a coordinate to each object, indicating its distance from a reference end of the target molecule. This paper describes a computational method and a body of software for assigning coordinates to map objects, given a solution or partial solution to the ordering problem. We describe our method in the context of multiple-complete-digest (MCD) mapping, but it should be applicable to a variety of other mapping problems. Because of errors in the data or insufficient clone coverage to uniquely identify the true ordering of the map objects, a partial ordering is typically the best one can hope for. Once a partial ordering has been established, one often seeks to overlay a metric along the map to assess the distances between the map objects. This problem often proves intractable because of data errors such as erroneous local length measurements (e.g., large clone lengths on low-resolution physical maps). We present a solution to the coordinate assignment problem for MCD restriction-fragment mapping, in which a coordinated set of single-enzyme restriction maps are simultaneously constructed. We show that the coordinate assignment problem can be expressed as the solution of a system of linear constraints. If the linear system is free of inconsistencies, it can be solved using the standard Bellman-Ford algorithm. In the more typical case where the system is inconsistent, our program perturbs it to find a new consistent system of linear constraints, close to those of the given inconsistent system, using a modified Bellman-Ford algorithm. Examples are provided of simple map inconsistencies and the methods by which our program detects candidate data errors and directs the user to potential suspect regions of the map.     

Molecular biological characterization of adenovirus DNA

The agarose gel electrophoresis of DNA, the ethidium bromide fluorescence detection of DNA fragments and restriction endonucleases were discovered at the end of the '60s. The methodological progress enabled institutions equipped with less sophisticated technology to achieve also unique experimental and scientific results in the field of viral DNA research. The team working on virus DNA within the adenovirus research group has constructed several new restriction endonuclease maps of the genomes of human and animal adenoviruses; contributed to the methodology of the determination of specific endonuclease sites, and genome polarity; discovered new restriction endonucleases, adenovirus subtypes, new empty capsid, and genome subpopulations; participated in cooperations leading to novel, although hypothetical approaches in AIDS therapy, taxonomic definition of viruses, and evolutionary origins of adenovirus replication and encapsidation strategy.     

Mapping the chicken genome: problems and perspectives

Various molecular methods are now used to map the chicken genome, including chromosome scraping, flow cytofluorimetry, zonal centrifugation, construction of chromosome-specific libraries, genetic analysis with polymorphic DNA markers, and in situ hybridization. Two main drawbacks are characteristic of existing maps of chicken chromosomes. First, classic genetic maps (i.e., linkage groups of genes for morphological, physiological, and biochemical characters), physical maps of chromosomes, and new genetic maps constructed on the basis of polymorphic DNA markers (RFLP, RAPD, VNTR, SSR, and CR1-PCR) do not coordinate with one another. Second, a relatively low number of genes is present in classic genetic maps and physical chromosome maps. Application of cytogenetic methods to chromosome mapping in birds is limited because of some specific features characteristic of the organization of avian genomes. For the same reason, studying the location and expression of avian genes is very important. Since mammalian and avian genomes differ in structure, revealing their possible common functional characteristics will provide for a better understanding of the general mechanisms that control biologically important characters in higher animals.     

Computer technology for genogenographic study of the gene pool. V. Evaluation of the reliability of maps

An important aspect of gene geography, the estimation of the reliability of interpolation maps, is considered. The introduced quantitative parameter, map reliability, was estimated as the probability of predicted character values in interpolated map regions. The obtained estimates characterize the statistic significance of the mapped values of the character. The estimation algorithm is based on concepts and mathematical methods of the reliability theory. The proposed approach involved the estimation of the reliability at each point of the mapped area and resulted in a new map (a reliability map) expressing the reliability of gene geographic mapping in probability terms. Approaches to estimate the reliability of mapping, as dependent on various parameters of the initial data, were proposed, a general computer-based technology was elaborated, and a standardized reliability scale was proposed. Reliability maps are considered necessary for the correct interpretation of gene geographic maps. The estimation of reliability as dependent on the number and distribution of initially tested populations was illustrated by the example of frequency maps of individual genes (HP*1, HLA*A1) and synthetic maps (100 alleles of 34 polymorphic loci) of Eastern Europe.     

Cerebral cartography--a method for visualizing cortical structures

We present a method for visualizing the human cortex on one planar map. The data are taken from a 3D MRI study. Ray tracing with non-parallel rays is used to project the cortical relief onto a non-planar projection surface, which is in turn mapped onto the plane by cartographical projection. Two modifications of the method are proposed: the spherical mapping uses a sphere as the projection surface; the model-based mapping uses an analytically defined model of the scalp to generate the normal vectors. The cerebral cartography can be used for example for producing anatomical reference maps on which EEG measurement data can be superimposed.     

NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.