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A clamp ligation method for point mutational spectrometry : marked increase in scanning range for the human genome

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dc.contributor.advisor William G. Thilly. en_US
dc.contributor.author Kim, Andrea Seungsun, 1971- en_US
dc.contributor.other Massachusetts Institute of Technology. Division of Bioengineering and Environmental Health. en_US
dc.date.accessioned 2008-11-10T20:01:29Z
dc.date.available 2008-11-10T20:01:29Z
dc.date.copyright 2002 en_US
dc.date.issued 2002 en_US
dc.identifier.uri http://dspace.mit.edu/handle/1721.1/8306 en_US
dc.identifier.uri http://hdl.handle.net/1721.1/8306
dc.description Thesis (Ph. D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2002. en_US
dc.description Includes bibliographical references (leaves 176-200). en_US
dc.description.abstract The study of human mutagenesis requires methods of measuring somatic mutations in normal human tissues and inherited mutations in human populations. Such methods should permit measurement of rare mutations in the presence of abundant wild-type copies and should be general to the human genome. A sensitivity of 2 x 10-6 for point mutations was recently achieved in human cells using a novel method of target isolation, constant denaturant capillary electrophoresis (CDCE), and high-fidelity polymerase chain reaction (hifi-PCR) (Li-Sucholeiki and Thilly, 2000). This method is applicable to 100-base pair (bp) DNA domains juxtaposed with a naturally occurring domain of a higher melting temperature, or a natural clamp. Such sequence domains represent about 9% of the human genome. To permit analysis of rare point mutations in the human genome more generally, this thesis developed a procedure in which a clamp can be ligated to any 100-bp sequence of interest. This procedure was combined with the previous method to create a new method of point mutational analysis that is not dependent on a naturally occurring clamp. To demonstrate the new method, a sequence with a natural clamp, a part of the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene (cDNA-bp 223-318), was analyzed using both the natural and ligated clamps. A sensitivity of 2 x 10-5 in human cells was demonstrated using the ligated clamp as opposed to 5 x 10-6 using the natural clamp. en_US
dc.description.abstract (cont.) The sensitivity of the new method using the ligated clamp was demonstrated to be limited by the fidelity of Pfu DNA polymerase used for PCR. The sequence of the ligated clamp accounted for the differences in sensitivity as a result of causing a decreased efficiency of mutant enrichment by CDCE. The new method can be applied to measure somatic mutations in normal human tissues, such as lung tissues, in which point mutations at fractions above 10-5 have been observed. This method can also detect predominant inherited mutations even for genes carrying recessive deleterious alleles in pooled samples derived from a large number of individuals. en_US
dc.description.statementofresponsibility by Andrea Seungsun Kim. en_US
dc.format.extent 200 leaves en_US
dc.language.iso eng en_US
dc.publisher Massachusetts Institute of Technology en_US
dc.rights M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. en_US
dc.rights.uri http://dspace.mit.edu/handle/1721.1/8306 en_US
dc.rights.uri http://dspace.mit.edu/handle/1721.1/7582 en_US
dc.subject Division of Bioengineering and Environmental Health. en_US
dc.title A clamp ligation method for point mutational spectrometry : marked increase in scanning range for the human genome en_US
dc.type Thesis en_US
dc.description.degree Ph.D. en_US
dc.contributor.department Massachusetts Institute of Technology. Division of Bioengineering and Environmental Health. en_US
dc.identifier.oclc 50445036 en_US


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