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

Duell, Thomas; Wang, Mei; Wu, Jihong; Kim, Ung-Jin

doi:10.1006/geno.1997.4954

Cited by 16 publications

(23 citation statements)

References 31 publications

(11 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All measurements were performed in triplicate; the results provided in the form of lists were imported into spreadsheets (MS Excel, Microsoft Corp., Redmond, WA) for further calculations. We used a factor of 2.3 kb/ μm for the conversion of the length of fibers or hybridization domains (measured in micrometers) to DNA fragment sizes (reported in kb) [16][17][18].…”

Section: Microscopy and Image Analysismentioning

confidence: 99%

“…Following hybridization and prior to triple color antibody detection, the slides were washed in 2X SSC (pH 7.0) and blocked with 100 μl PN buffer (0.1 M sodium phosphate buffer, pH 8.0, 1% Nonidet-P40 (Sigma)) supplemented with 5% non-fat milk powder (Carnation, Wilkes-Barre, PA) [37]. Detection was carried out as described [16][17][18]. Slides were mounted in 1% p-phenylenediamine antifade solution (Sigma) in glycerol [38] and stored at −20°C.…”

Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

“…Using 'Quantitative DNA Fiber Mapping' (QDFM) which allows the delineation of deletions and clone overlaps by direct visualization, we constructed high resolution physical maps for several regions of the human genome, among them a chromosome 22-specific region covered by three different sub-clones of an unstable YAC clone [16][17][18][19][20]. In a nutshell, QDFM is based on the hybridization of probe DNA prepared from one or several clones onto stretched DNA molecules isolated from another, often larger clone, such as a YAC clone or even genomic DNA [16].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Greulich-Bode¹,

Wu²,

Duell³

2009

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Microscopy and Image Analysismentioning

confidence: 99%

Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Greulich-Bode¹,

Wu²,

Duell³

2009

Self Cite

View full text Add to dashboard Cite

show abstract

“…3. The YAC cloning vectors pJs97 and pJs98, cloned in plasmid vectors (Invitrogen), can be used to prepare probes useful to determine the orientation of the YAC insert (56). For this purpose, plasmid DNA is extracted using the above alkaline lysis protocol or a commercial kit and labeled by random priming as described below.…”

Section: Generation Of Probes By In Vitro Dna Amplificationmentioning

confidence: 99%

“…Macintosh, IBM/PC or SUN computers that allows the user to trace DNA fibers by drawing a straight or segmented line and then calculates the length of the line in pixels (52,56). The pixel spacing for the camera and the microscope objective used 26 in the experiment (a 63x objective is used for molecules up to 100 kb, a 40x objective for larger molecules) is known and is converted into µm or into kb using the factor of 2.3 kb/µm (51).…”

Section: For Determination Of Map Positions Interactive Software Is mentioning

confidence: 99%

Quantitative DNA Fiber Mapping in Genome Research and Construction of Physical Maps

Chu

Gene Mapping, Discovery, and Expression

Self Cite

View full text Add to dashboard Cite

Efforts to prepare a first draft of the human DNA genomic sequence forced multidisciplinary teams of researchers to face unique challenges. At the same time, these unprecedented obstacles stimulated the development of many highly innovative approaches to biomedical problem solving, robotics and bioinformatics. High resolution physical maps are required for ordering individual segments of information for the construction of a comprehensive map of the entire genome. This article describes a novel way to identify, delineate and characterize selected, often small DNA sequences along a larger piece of the human genome. The technology is based on immobilization of high molecular weight DNA molecules on a solid substrate (such as a glass slide) followed by uniform stretching of the DNA molecule by the force of a receeding meniscus. The hydrodynamic force stretches the DNA molecules homogeneously to approximately 2.3 kb/μm, so that distances measured after probe binding in μm can be converted directly into kb distances. Out of a large number of applications, this article focusses on mapping of genomic sequences relative to one another, the assembly of physical maps with near kilobase (kb) resolution and finally, quality control during physical map assembly and sequencing.

show abstract

Optical Mapping in Genomic Analysis

Aston

Schwartz

2000

Encyclopedia of Analytical Chemistry

View full text Add to dashboard Cite

Optical mapping is a system for the construction of ordered restriction maps from single deoxyribonucleic acid (DNA) molecules. DNA molecules bound to derivatized glass surfaces are cleaved with restriction enzymes and imaged by fluorescence microscopy. Cut sites are visualized as gaps between cleaved DNA fragments, which retain their original order. Optical mapping offers advantages over electrophoretic methods of restriction enzyme analysis, which require separate techniques for separating cleaved DNA fragments and determining fragment order. Furthermore, small amounts of DNA are required for optical mapping analysis. By determining the existence of these sequence‐specific cut sites and the distances between them, a precise landmark map of the DNA sequence can be constructed. Prokaryote and eukaryote DNA can be mapped. Maps can be constructed from both cloned DNA and genomic DNA. Restriction maps can be constructed for whole genomes by overlapping maps derived from individual molecules. Such maps provide a useful scaffold for accurate alignment of sequence contigs generated by a shotgun sequencing approach and a means of comparing populations and strains.

show abstract

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

Cited by 16 publications

References 31 publications

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

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

Quantitative DNA Fiber Mapping in Genome Research and Construction of Physical Maps

Optical Mapping in Genomic Analysis

Contact Info

Product

Resources

About