Double-Tagging Ligation for Library Construction of Double- Strand Break Regions in Sperm Chromatin Fragmentation

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2015-05
Authors
Jin, Xiao
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[Honolulu] : [University of Hawaii at Manoa], [May 2015]
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Sperm chromatin fragmentation (SCF) (Shaman et al. 2006) is a chemical induction of endogenous DNA fragmentation including both double-strand and single-strand breaks in mouse spermatozoa. The reaction can be activated by Mn (II) alone or in concert with Ca (II). The resultant double-strand breaks (DSBs) occur at a regular 25 kb interval throughout the mouse sperm genome. It has been postulated that the SCF-induced DSB regions (SCFRs) may be associated with the nuclear matrix attachment regions (MARs) in the mouse spermatozoa (Ward 2010). Such proposition demands a technical solution in sequencing of the SCFRs and subsequent mapping of the reads to the mouse genome. In this study, We have developed a double-tagging ligation protocol for sequencing the SCFRs. We first purified the SCF fragments and labeled the SCFRs. We then enclosed the SCFRs into the final sequencing construt containing sequencing adaptors on each end. The library preparation for SFCR break site analysis that we have performed in this study is a technical quest necessary for understanding and characterization of these structurally unique DNA elements.
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M.S. University of Hawaii at Manoa 2015.
Includes bibliographical references.
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Theses for the degree of Master of Science (University of Hawaii at Manoa). Anatomy, Biochemistry & Physiology
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