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Rapid method for construction of yeast artificial chromosome human DNA libraries involving the trapping of cells in agarose films
Author(s) -
Kai Tatsuo,
Song Jac-chan,
Tashiro Hiroyuki,
Saka Fujiko,
Ozawa Kazuo,
Miwa Tokiko,
Kitabayashi Issay,
Soeda Ei-ichi,
Yokoyama Kazushige
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)81443-r
Subject(s) - agarose , yeast artificial chromosome , dna , genomic library , microbiology and biotechnology , in vitro recombination , biology , cloning (programming) , cloning vector , genomic dna , molecular cloning , transformation (genetics) , library , human genome , yeast , genetics , chromosome , recombinant dna , gene , vector (molecular biology) , genome , gene mapping , base sequence , computer science , gene expression , programming language , 16s ribosomal rna
A simple method for molecular cloning of fragments of more than one hundred kilobase pairs of exogenous DNA, by the encapsulation of cell in agarose beads, was reported previously for the construction of a human genomic DNA library in a yeast artificial chromosome (YAC) vector in situ YAC construction [1]. The efficiency of this procedure is impaired by the step in which agarose beads that contain human DNA fragments are melted before transformation. The incomplete solubility of the ligated human DNA fragment. YAC vector often results in lower than desirable frequencies of transformation. To overcome this problem we have developed a new improved method that involves use of an agarose film. The technical manipulations involved in the construction of clones of very large segments of human DNA are discussed.