Rapid method for construction of yeast artificial chromosome human DNA libraries involving the trapping of cells in agarose films

Abstract

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.

Single DNA marker generated by “YAC-Alu PCR” that is end-specific

“…Yeast cells were embedded and lysed in agarose (agarose plugs) by the method of Carle and Olson (1984). High-molecular-weight DNA was extracted from yeast colonies and YAC recombinant clones as described elsewhere (Carle et al, 1984;Yokoyama et al, 1990;Kai et al, 1990;Imai and Olson, 1990 EagI or EcoRI, 10 ug of DNA were subjected to electrophoresis on a 1 ~ agarose/ Tris-acetate gel and transferred to a nylon membrane. Gels were treated with 0.25 N HC1 for 15 rain, then with a solution of 0.4 N NaOH and 0.6 M NaC1 for 15 min, and finally with a solution of 1.5 M NaC1 and 0.5 M Tris-HC1 (pH 7.5), before transfer to nylon membranes, as described elsewhere (Carle et al, 1986), and subsequent crosslinking (Church and Gilbert, 1984).…”

“…The YAC library specific for human chromosome 21 DNA had been prepared previously, as described in detail elsewhere . In brief, the DNA from 153E9a3 cells were entrapped in agarose beads or agarose films as described Kai et al, 1990). We estimated that there were approximately 50 ~g of genomic DNA per ml of agarose beads (or films) that contained approximately 8 x 106 cells.…”

“…High-molecular-weight DNA was digested with EagI or NotI and ligated to the arms of pYAC55 to yield products with an average size of 150 to 160 kb. Screening of yeast colonies with a probe for human Alu DNA was performed as described previously Kai et al, 1990).…”

“…Construction of a yeast artificial chromosome (YAC) library specific for human chromosome 21 was carried out by a new method that involves entrapping cells in agarose beads (or agarose film) and digesting the DNA with the restriction enzymes Notl or Eagl Kai et al, 1990). Approximately 1.0 pg of DNA from the somatic hybrid cell line 153E9a3 generated 2-4 x 108 transformants.…”

Yeast artificil chromosome (YAC) clones and sequence tagged site (STS) markers anchored at human chromosome 21

“…Yeast artificial chromosome (YAC) vector developed recently provides a powerful tool for cloning several hundred kilobases of exogenous DNA in yeast cells and has allowed us to analyze a large region of human genome (Burke et al, 1987;Hieter et al, 1990;Imai and Olson, 1990a;Kai et al, 1990;Yokoyama et al, 1990).…”