Transposon based mutagenesis and mapping of transposon insertion sites within the Ehrlichia chaffeensis genome using semi random two-step PCR

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dc.contributor.author Indukuri, Vijaya Varma
dc.date.accessioned 2013-08-22T19:13:15Z
dc.date.available 2013-08-22T19:13:15Z
dc.date.issued 2013-08-22
dc.identifier.uri http://hdl.handle.net/2097/16329
dc.description.abstract Ehrlichia chaffeensis a tick transmitted Anaplasmataceae family pathogen responsible for human monocytic ehrlichiosis. Differential gene expression appears to be an important pathogen adaptation mechanism for its survival in dual hosts. One of the ways to test this hypothesis is by performing mutational analysis that aids in altering the expression of genes. Mutagenesis is also a useful tool to study the effects of a gene function in an organism. Focus of my research has been to prepare several modified Himar transposon mutagenesis constructs for their value in introducing mutations in E. chaffeensis genome. While the work is in progress, research team from our group used existing Himar transposon mutagenesis plasmids and was able to create mutations in E. chaffeensis. Multiple mutations were identified by Southern blot analysis. I redirected my research efforts towards mapping the genomic insertion sites by performing the semi-random two step PCR (ST-PCR) method, followed by DNA sequence analysis. In this method, the first PCR is performed with genomic DNA as the template with a primer specific to the insertion segment and the second primer containing an anchored degenerate sequence segment. The product from the first PCR is used in the second PCR with nested transposon insertion primer and a primer designed to bind to the known sequence portion of degenerate primer segment. This method aided in identifying the genomic locations of four E. chaffeensis mutants and also was valuable in confirming four other sites mapped previously by the rescue cloning method. This is the first mutational analysis study in the genome of an Ehrlichia species. Mapping the genomic transposon insertion sites is the first critical step needed for the continued research to define the importance of the mutations in understanding the pathogenesis caused by the organism. en_US
dc.description.sponsorship This research project is funded by the grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA (AI070908). en_US
dc.language.iso en_US en_US
dc.publisher Kansas State University en
dc.subject Transposon mutagenesis en_US
dc.subject Mapping en_US
dc.subject Insertion mutations en_US
dc.subject Ehrlichia chaffeensis en_US
dc.title Transposon based mutagenesis and mapping of transposon insertion sites within the Ehrlichia chaffeensis genome using semi random two-step PCR en_US
dc.type Thesis en_US
dc.description.degree Master of Science en_US
dc.description.level Masters en_US
dc.description.department Department of Diagnostic Medicine/Pathobiology en_US
dc.description.advisor Roman Reddy Ganta en_US
dc.subject.umi Molecular Biology (0307) en_US
dc.date.published 2013 en_US
dc.date.graduationmonth August en_US


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