Base sequence studies of 300 nucleotide renatured repeated human DNA clones

Encapsulation of cells in agarose beads for use in the construction of human DNA libraries as yeast artificial chromosomes (YAC)

“…Under optimal transformation conditions, the transformation of yeast spheroplasts generated approximately 10 a clones per microgram of DNA. The total genomic DNAs of the randomly chosen ura § trp +, ade-transformants were fractionated on PFGE, blotted on the nitrocellulose and hybridized with a radiolabeled human repetitive Alu sequence probe (Deininger et al, 1981), and with radiolabeled pBR322 and human genomic DNA probes. One of these analyses is shown in Fig.…”

“…A pilot screening experiment was performed using an ordered 96-well array of tested clones. The probes used to identify human cloned genomic DNA were a 280-bp BamHI fragment of BLUR-8 (Alu probe); (Deininger et al, 1981), pBR322, and human placenta genomic DNA. The probes were radiolabeled with [32P]dCTP using an oligopriming labeling kit (Pharmacia-LKB, Bromma) as described by Feinberg and Volgelstein (1983).…”

Multicolor fluorescence in situ hybridization on metaphase chromosomes and interphase Halo-preparations using cosmid and YAC clones for the simultaneous high resolution mapping of deletions in the dystrophin gene

“…With the emergence of interspersed repeat sequence PCR protocols (Nelson et al 1989;Ledbetter et al 1990) that selectively amplify the human content in a background of, for example, hamster hybrid cell lines (Lengauer et al 1990;Lichter et al 1990), YAC clones became suitable probes for FISH in diagnostic settings. Alu repeats (Deininger et al 1981) are the most common repeat sequences in man, with a copy number of about 10 6 . This results in an average distance of the repeat of about 4 kb (for a review, see Kariya et al 1987).…”

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

“…Hybridization ,,va,~ carried out iri a solution of I M NaCI, 10% dextraa sulfate and 1,0% sodium dodecyl sulfate with 100 tLg/ml of yeast tRNA as carrier and approxirnately I0 c' cpnl/ml of "~P-labeled probe, 'rile probes used were a 280-bp BamHl fragmerit of BLUR-8 (AIu probe) [12] and Pvull/BumHl digests of pBR 322, radiolabeled with [~P]dCTP v,,ith an oligopriming labeling kit (Pharmacia-LKB) [13]. Membranes were washed twice with 2 x SSC at room temperature for 10 mhl, then once with 2 x SSC, 1,0°70 SDS at 65°C once for 30 mix, and finally once with 0.1 x S$C at room temperature once for 30 mix, Autoradiography was performed at --80°C for a day.…”