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Identification and isolation of Mu-flanking fragments from maize

摘要Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics.Isolation of genomic seg-ments flanking the insertion Mutator (Mu) is a key step in insertion mutagenesis studies.Herein,we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize.The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl ⅡMsp Ⅰ and ligation of digested fragments to the Bgl Ⅱ- and Msp Ⅰ-adaptors; 2) enrichment of a subset of Bgl Ⅱ/Msp Ⅰ fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking re-gions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions.Using this approach,we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-indueed mutant,and one such fragment,M196-FF,is found to contain a partial sequence of the DNA topoisomerase Ⅰ gene Topl.Moreover,the modified AFLP method including all restriction enzymes,adaptors and primers has been optimized in this study.The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.

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分类号 Q3
栏目名称 Research Reports
DOI 10.1016/S1673-8527(08)60029-6
发布时间 2008-06-02
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