|
| |
|
Most current tests for human exposure to environmental mutagens are only indicators of genetic damag ... - Aaron Hall |
| |
|
Knowledge without wisdom can lead to disaster. A degree minus common sense will not add up to succes ... - Lynn Bradley |
| |
|
Are features offered by Dish TV making its Satellite TV service a valuable alternative? - dave4 |
| |
|
|
| |
|
Distance Learning allows students to take college courses at times and places that meet the demands ... - Michael Bustamante |
| |
|
In his presentation at the 1998 Cambridge meeting, James Wilson characterized gene therapy as a nove ... - Aaron Hall |
| |
|
I offer these as a metaphysical exposition of the basic dimensions defining space/time as we experie ... - LariAnn Garner |
| |
|
Cancer is a life-threatening disease that is prevalent in our time. One of which is known as non-Hod ... - Morgan Hamilton |
| |
|
Development of cheaper and faster technologies for large-scale genome mapping has been a major prior ... - Aaron Hall |
| |
|
| |
|
|
Development of cheaper and faster technologies for large-scale genome mapping has been a major priority in the first 5 years of the Human Genome Project. Although many efforts have focused on improving standard gel electrophoresis and hybridization methods,a new approach using optical detection of single DNA molecules shows great promise for rapid construction of ordered genome maps based on restriction endonuclease cutting sites.(1-4)
Restriction endonucleases enzymes that cut DNA molecules at specific sites in the genome have played a major role in allowing investigators to identify and characterize various loci on a DNA molecule. Unlike maps based on STSs (a sequence-based landmark), restriction maps provide the precise genomic distances that are essential for efficient sequencing and for determining the spatial relationships of specific loci. Compared with hybridization-based fingerprinting approaches, ordered restriction maps offer relatively unambiguous clone characterization, which is useful for determining overlapping areas in contig formation, establishing minimum tiling paths for sequencing (coverage of a region), and characterizing genetic lesions with respect to various structural alterations.
Despite the broad applications of restriction maps, however, associated techniques for their generation have changed little over the last 10 years because of their reliance on tedious electrophoresis methods. Optical mapping of single DNA molecules represents the first practical nonelectrophoretic genomic-analysis approach toward producing ordered restriction maps.
Visualizing Gaps in a DNA Molecule
Ordered optical restriction maps were first constructed from yeast chromosomes by fluorescence microscopic imaging of stained DNA molecules treated with restriction enzymes(1).In this method, individual fluorescently labeled DNA molecules were elongated on a microscope slide in a molten agarose flow containing restriction endonucleases. Resulting cleavage events were recorded by fluorescence microscopy as time-lapse digitized images; cut sites appeared as gaps that widened as DNA fragments relaxed. Fragment order was apparent throughout the procedure, and maps were constructed by measuring fragment sizes via relative fluorescence intensity or apparent length measurements. In addition to high throughput and high resolution, advantages of optical mapping include a very small sample size and the elimination of radioactive labeling required in conventional methods.
Modifications for Other Vectors
Improvements to the original optical mapping method now allow analysis of a wide range of such cloning vectors as cosmid, bacteriophage, P1, and YACs and produce accurate maps consisting of DNA fragments as small as 500 bp. These improvements include eliminating agarose and time-lapse imaging and fixing the elongated DNA molecules onto polylysine-treated glass surfaces. To analyze lambda clones, DNA samples have been fixed onto derivatized glass surfaces by sandwiching them between a treated coverslip and glass slide. A cooled CCD camera was used to image molecules from 28 kb down to 800 bp(3); more recent experiments have lowered the resolution limit to about 300 bp. Sizing errors are comparable to and in many cases lower than the rate achievable by agarose gel electrophoresis, depending on the number of molecules analyzed.
Generating YAC Maps
Although a large fraction of the human genome is covered by YAC contigs, few YAC restriction maps have been generated. Using optical mapping, ordered YAC restriction maps have been constructed, (4) with overall relative sizing errors comparable to routine pulsed-field gel electrophoretic analysis. Ordered restriction maps have now been generated for the human Beckwith-Wiedeman locus [with David Housman (Massachusetts Institute of Technology)], the BRCA2 locus [with Stuart Fisher (Columbia University)], and the mouse olfactory locus [with Richard Axel (Columbia University)]. Optical maps are currently being generated from phage, cosmid, YAC, and BAC clones.
Large-Scale Genome Mapping
High-throughput approaches are being devised in anticipation of the vastly increased requirements for whole-genome analysis. Fully automated optical mapping approaches would require no human intervention between sample preparation and map construction and hold enormous promise for miniaturization. The advantages of optical mapping high throughput and resolution, safety, and low cost are likely to aid rapid progress in genome analysis and contribute significantly to the accelerating pace of the Human Genome Project as well as to efforts directed toward mapping human disease genes and other genetic alterations. |
| Author: Aaron Hall |
| |
Author Bio:
|
|
 |
|
| |
Related Articles |
| |
Hudson Heads NCHGR Policy Office
|
| |
Tracing Patterns in Nature Backwards
|
| |
The Perfect Way To Bearcat Scanner Uniden
|
| |
Technology and the Pace of Change in our Lives is Becoming More Rapid
|
| |
How To Auto Security System For Your Car
|
| |
HGP Grantees Report Progress, Challenges
|
| |
Drosophila Sequencing Nears Completion
|
| |
Static Charge Pulse on Wind Generator Material Memory Blades
|
| |
Business Technology Schools
|
| |
"Human Gene Therapy: Present and Future'
|
| |
|
|
|
|
Main
