A system of rapid isolation of end-DNA from a small amount of fosmid DNA, with vector-based PCR for chromosome walking.
The pBAC 108L and pFos 1 vectors were developed as stable propagation vectors which, due to their extremely low copy number, facilitate the cloning of a large-sized insert containing repeated DNA. However, the low copy number requires laborious end-DNA preparation for end sequencing and chromosome walking. Here we describe efficient methods for end-DNA isolation. The entire process, including small-scale DNA preparation, restriction digestion, self-ligation, and PCR with vector-based primers, is carried out in 96-well formats. Using a Fosmid library of genomic DNA of Candida albicans, PCR products ranging in size from 0.1 to 8 kbp were generated from 118 end sequences in 140 reactions from 70 Fosmid clones. A single or a prominent band was found in 101 of these reactions. Twenty-six of these bands were tested for walking and all of them proved to be specific. Thus, the system overcomes the disadvantage caused by low copy number. This system allows rapid physical mapping of genomes, and is adaptable for several other vectors including BAC (bacterial artificial chromosome), PAC (P1-derived artificial chromosome), and YAC (yeast artificial chromosome).
Chibana
H
H
Department of Genetics, Cell Biology, and Development, University of Minnesota, St. Paul 55108, USA. chibana@biosci.cbs.umn.edu
Heinecke
E L
EL
Beckerman
J L
JL
Magee
P T
PT
D001483:Base Sequence; D002176:Candida albicans / Q000235:genetics*; D016386:Chromosome Walking / Q000379:methods*; D022201:Chromosomes, Artificial / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction / Q000379:methods
D017931:DNA Primers; D004271:DNA, Fungal